Fluorinated resorufin compounds and their application

ABSTRACT

The invention provides novel fluorinated resorufin compounds that are of use in a variety of assay formats. Also provided are methods of using the compounds and kits that include a compound of the invention and instructions detailing the use of the compound in one or more assay formats.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 12/015,476, filedJan. 16, 2008, which is a divisional application of U.S. Ser. No.10/980,139, filed Nov. 1, 2004 (now U.S. Pat. No. 7,432,372), whichapplication claims priority to U.S. Ser. No. 60/516,244, filed Oct. 31,2003, which disclosures are incorporated herein by reference in theirentirety.

FIELD OF THE INVENTION

The present invention relates to novel fluorogenic compounds that haveutility in detecting reactive oxygen species, e.g., hydrogen peroxide.The invention is of use in a variety of fields including immunology,diagnostics, molecular biology and fluorescence based assays.

BACKGROUND OF THE INVENTION

The study and detection of enzyme activity serve a wide range ofpurposes both in research laboratories and in clinical assays. Enzymeactivity is monitored, for example, in determining physiologicalfunctions in patients during routine checkups or diagnostic proceduresin general, in monitoring the exposure of workers and others topotentially harmful chemicals such as toxic or carcinogenic pesticidesor inorganic materials in the atmosphere, soil, or drinking water, indetermining the effectiveness of pharmaceuticals on disease states orconditions, in screening new compounds for biological activity as eitherpromoters or inhibitors of particular enzymes, in monitoring gene andtransgene expression, and in performing immunological and otherlaboratory assays such as enzyme-linked immunosorbent assays (ELISAs)and Western blots.

Optical methods of detection, such as fluorescence emission, UVabsorptivity, and colorimetry are convenient and highly effective fordetecting, monitoring, and measuring enzyme activity, since methods suchas these can generate either qualitative or quantitative information anddetection can be achieved either by direct visual observation or byinstrumentation. Optically detectable reporters, i.e., synthetic orsubstitute substrates that are added to a sample and that display ameasurable increase or other difference in optical detectability uponaction of the enzyme, are therefore particularly useful. Examples ofoptical reporters that are currently known are 4-nitrophenol,α-naphthol, β-naphthol, resorufin and substituted resorufins,nitranilide, 5-bromo-4-chloro-3-indole, coumarin, xanthene andumbelliferone derivatives. The degree of change and hence theeffectiveness of optical detection reporters depend on any of severalfactors, depending on the detection method for which they are used. Someof these factors are, a high extinction coefficient for reporters thatare detectable by light absorptivity (particularly a large increase fromsubstrate to product), a large change in the wavelength at which maximumabsorptivity occurs (particularly a large substrate-to-product redshift), a substrate-to-product increase in the Stokes' shift forfluorescent reporters, and the chemical stability of the reporter.

With the advent of nanotechnology, there is an increased ability toperform numerous chemical and physical operations with very smallvolumes. This opportunity comes with the requirement that determinationshave enhanced sensitivity to detect the few molecules that are presentto provide the detectable signal. Part of the increased sensitivity maycome from more sensitive detectors, but these are usually more expensiveand are not readily available in most laboratories. An alternative isthe provision of assays that rely on readily detectable labels. Theassays may also be formatted to use compounds that are readily acceptedby an enzyme as a substrate and efficiently convert a fluorogenicsubstrate to a fluorescent label.

Due to their reliable oxidation/reduction chemistry, resorufins areattractive fluorogenic substrates for use in assays to detect reactiveoxygen species, e.g., peroxides, or enzymes that generate such species,e.g., peroxidases. Many resorufins are known in the art. For example,Miike et al. (U.S. Pat. Nos. 4,384,042; and 4,954,630) disclose the useof resorufins to detect hydrogen peroxide. Klein et al. (U.S. Pat. No.5,304,645) discuss the preparation and use of a series of reactiveresorufin derivatives and their conjugation to species such as ligands,haptens, antigens, antibodies and the like. Mühlegger et al. (U.S. Pat.No. 4,719,097) set forth resorufin phosphates for determining theactivity of phosphatases. None of the cited references discloses afluorinated resorufin analogue such as those of the present invention.Furthermore, until the present invention, the safe and reliablepreparation of fluorinated resorufin derivatives was not known in theart.

SUMMARY OF THE INVENTION

It has now been discovered that fluorinated resorufins can be safely andreliably prepared. The fluorinated derivatives have improved fluorescentproperties relative to non-fluorinated resorufin species. For example,the fluorescence of fluorinated resorufins is much more stable in highconcentration of peroxide than the non-fluorinated resorufins. Due toits lower pKa, fluorination of the phenoxazine ring system of theresorufin markedly enhances the fluorescence intensity of thecorresponding resorufin. The fluorinated resorufins are also morephotostable under prolonged irradiation than the non-fluorinatedanalogues.

Thus, in a first aspect, the present invention provides a compoundhaving the general formula:

in which the symbol R^(a) represents H, substituted or unsubstitutedalkyl, substituted or unsubstituted aryl, O, and C(X^(a))R^(a1), inwhich X^(a) represents O, S or NH and R^(a1) is a member selected fromsubstituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl,OR^(a2) and NR^(a3)R^(a4). The symbols R^(a2), R^(a3) and R^(a4)independently represent moieties such as H, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted heteroaryl and substituted or unsubstitutedheterocycloalkyl. The index “n” represents either 0 or 1. R^(b), R^(c),R^(e), R^(f), R^(h) and R^(i) are members that are independentlyselected from the genus of aryl substituents, including species such asH, OH, sulfo, nitro, carboxyl, carboxylate esters, halogen, substitutedor unsubstituted alkyl, substituted or unsubstituted heteroalkyl (e.g.,alkoxy, alkylthio, aminoalkyl, etc.), substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, substituted or unsubstitutedheterocycloalkyl and a reactive group. At least one of R^(b), R^(c),R^(e), R^(f), R^(h) and R^(i) is fluorine.

The symbol R^(d) represents OR^(d1) or NR^(d1)R^(d2). R^(d1) and R^(d2)are members independently selected from H, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted acyl, and a reactive group. R^(g)represents OR^(g1), NR^(g1)R^(g2) or (═O). The identities of R^(g1) andR^(g2) are the same as those set forth above for R^(d1) and R^(d2).

The invention also provides methods for using the fluorinated compoundsto assay samples for the presence of a reactive oxygen species, such asperoxide, for detection of a specific analyte and for measuringmetabolic activity in a cell. In an exemplary embodiment, the assay isof use to detect and/or quantitate a reactive oxygen species or anenzyme that generates a reactive oxygen species in the sample.

In still a further aspect, the invention provides kits that include acompound of the invention and directions for making use of the compound.

Other aspects, objects and advantages of the present invention will beapparent from the detailed description that follows.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: is a plot of the fluorescence of Amplex Red reagent vs. Compound4 in a high hydrogen peroxide titration in a horseradish peroxidaseassay. Initially the fluorescence intensity of both dyes is relativelystable in the presence of high concentrations of H₂O₂, but after twentyminutes of incubation, there is a biphasic mode to the dilution series,whereby the fluorescence has a peak at ˜40 μM H₂O₂, then quickly dropsat 80-160 μM H₂O₂ rising slowly at higher concentrations.

FIG. 2: is a plot of a cycloxygenase assay comparing the fluorescence ofAmplex Red reagent with Compound 4. The plot shows that both dyereagents are oxidized to their fluorescent forms by COX-2. The dynamicrange and sensitivity of both dyes is similar with Compound 4demonstrating a greater fluorescent intensity signal. Error bars in thegraph are one standard deviation from the mean of three measurements.

FIG. 3: is a plot of a hemoglobin assay comparing the fluorescence ofAmplex Red reagent with Compound 4. The figure shows that both dyereagents are oxidized to their fluorescent forms by bovine hemoglobin.Although the dynamic range and sensitivity of both dyes is similar,Compound 4 is brighter. Error bars in the graph are one standarddeviation from the mean of three measurements.

FIG. 4: is a plot showing the time course of a glycerol assay comparingthe fluorescence of Amplex Red reagent with Compound 4. The figure showsthat both dye reagents are oxidized to their fluorescent forms by bovinehemoglobin. Although the dynamic range and sensitivity of both dyes issimilar, Compound 4 is brighter. Error bars in the graph are onestandard deviation from the mean of three measurements.

FIG. 5: is a plot comparing the relative fluorescent signal of AmplexRed reagent compared to Compound 4 in an ELISA for c-reactive proteinusing Goat anti-Rabbit IgG-HRP conjugate as a secondary antibody.

FIG. 6: is a graph showing the pH tolerance of Amplex Red reagent (6A)compared to the pH tolerance of Compound 4 (6B).

FIG. 7: is a graph showing the relative fluorescent signal generated byAmplex Red reagent compared to Compound 4 in an assay of LPS-inducedCOX-2 activity from RAW 264.7 cell lysate.

FIG. 8: is a graph showing relative fluorescence as a function ofphytase concentration. Error bars are one standard deviation. See,Example 41

FIG. 9: is a graph showing relative fluorescence as a function ofhorseradish peroxidase concentration with 50 μM hydrogen peroxide (finalassay concentration) at pH 5.5. Error bars are one standard deviation.See, Example 42.

FIG. 10: is a graph showing the limit of detection and dynamic range forthe detection of myeloperoxidase using Compound 4. See, Example 43.

DETAILED DESCRIPTION OF THE INVENTION Introduction

There is a continuous and expanding need for rapid, highly specificmethods of detecting and quantifying chemical, biochemical andbiological analytes in research and diagnostic mixtures. Of particularvalue are methods for measuring small quantities of nucleic acids,peptides (e.g., enzymes), pharmaceuticals, metabolites, microorganismsand other materials of diagnostic value. Examples of such materialsinclude narcotics and poisons, drugs administered for therapeuticpurposes, hormones, pathogenic microorganisms and viruses, antibodies,and enzymes and nucleic acids, particularly those implicated in diseasestates.

One method of detecting an analyte relies on directly or indirectlylabeling the analyte or other component of the analysis mixture with afluorescent species. Fluorescent labels have the advantage of requiringfew precautions in handling, and being amenable to high-throughputvisualization techniques (optical analysis including digitization of theimage for analysis in an integrated system comprising a computer).Preferred labels are typically characterized by one or more of thefollowing: high sensitivity, high stability, low background, lowenvironmental sensitivity and high specificity in labeling.

As discussed herein, the present invention provides a new class offluorinated fluorescent probes that are of use in a variety ofanalytical techniques.

DEFINITIONS

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to specific compositionsor process steps, as such may vary. It must be noted that, as used inthis specification and the appended claims, the singular form “a”, “an”and “the” include plural referents unless the context clearly dictatesotherwise. Thus, for example, reference to “a fluorogenic compound”includes a plurality of compounds and reference to “an enzyme” includesa plurality of enzymes and the like.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention is related. The following terms aredefined for purposes of the invention as described herein. The symbol

, whether utilized as a bond or displayed perpendicular to a bondindicates the point at which the displayed moiety is attached to theremainder of the molecule, solid support, etc.

Certain compounds of the present invention can exist in unsolvated formsas well as solvated forms, including hydrated forms. In general, thesolvated forms are equivalent to unsolvated forms and are encompassedwithin the scope of the present invention. Certain compounds of thepresent invention may exist in multiple crystalline or amorphous forms.In general, all physical forms are equivalent for the uses contemplatedby the present invention and are intended to be within the scope of thepresent invention.

Certain compounds of the present invention possess asymmetric carbonatoms (optical centers) or double bonds; the racemates, diastereomers,geometric isomers and individual isomers are encompassed within thescope of the present invention.

The compounds of the invention may be prepared as a single isomer (e.g.,enantiomer, cis-trans, positional, diastereomer) or as a mixture ofisomers. In a preferred embodiment, the compounds are prepared assubstantially a single isomer. Methods of preparing substantiallyisomerically pure compounds are known in the art. For example,enantiomerically enriched mixtures and pure enantiomeric compounds canbe prepared by using synthetic intermediates that are enantiomericallypure in combination with reactions that either leave the stereochemistryat a chiral center unchanged or result in its complete inversion.Alternatively, the final product or intermediates along the syntheticroute can be resolved into a single stereoisomer. Techniques forinverting or leaving unchanged a particular stereocenter, and those forresolving mixtures of stereoisomers are well known in the art and it iswell within the ability of one of skill in the art to choose andappropriate method for a particular situation. See, generally, Furnisset al. (eds.), VOGEL'S ENCYCLOPEDIA OF PRACTICAL ORGANIC CHEMISTRY5^(TH) ED., Longman Scientific and Technical Ltd., Essex, 1991, pp.809-816; and Heller, Acc. Chem. Res. 23: 128 (1990).

The compounds of the present invention may also contain unnaturalproportions of atomic isotopes at one or more of the atoms thatconstitute such compounds. For example, the compounds may beradiolabeled with radioactive isotopes, such as for example tritium(³H), iodine-125 (¹²⁵I) or carbon-14 (¹⁴C). All isotopic variations ofthe compounds of the present invention, whether radioactive or not, areintended to be encompassed within the scope of the present invention.

Where substituent groups are specified by their conventional chemicalformulae, written from left to right, they equally encompass thechemically identical substituents, which would result from writing thestructure from right to left, e.g., —CH₂O— is intended to also recite—OCH₂—.

The term “acyl” or “alkanoyl” by itself or in combination with anotherterm, means, unless otherwise stated, a stable straight or branchedchain, or cyclic hydrocarbon radical, or combinations thereof,consisting of the stated number of carbon atoms and an acyl radical onat least one terminus of the alkane radical. The “acyl radical” is thegroup derived from a carboxylic acid by removing the —OH moietytherefrom.

The term “alkyl,” by itself or as part of another substituent means,unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include divalent(“alkylene”) and multivalent radicals, having the number of carbon atomsdesignated (i.e. C₁-C₁₀ means one to ten carbons). Examples of saturatedhydrocarbon radicals include, but are not limited to, groups such asmethyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl,sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, homologsand isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, andthe like. An unsaturated alkyl group is one having one or more doublebonds or triple bonds. Examples of unsaturated alkyl groups include, butare not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl,2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and3-propynyl, 3-butynyl, and the higher homologs and isomers. The term“alkyl,” unless otherwise noted, is also meant to include thosederivatives of alkyl defined in more detail below, such as“heteroalkyl.” Alkyl groups that are limited to hydrocarbon groups aretermed “homoalkyl”.

Exemplary alkyl groups of use in the present invention contain betweenabout one and about twenty five carbon atoms (e.g. methyl, ethyl and thelike). Straight, branched or cyclic hydrocarbon chains having eight orfewer carbon atoms will also be referred to herein as “lower alkyl”. Inaddition, the term “alkyl” as used herein further includes one or moresubstitutions at one or more carbon atoms of the hydrocarbon chainfragment.

The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) areused in their conventional sense, and refer to those alkyl groupsattached to the remainder of the molecule via an oxygen atom, an aminogroup, or a sulfur atom, respectively.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a straight or branched chain, or cycliccarbon-containing radical, or combinations thereof, consisting of thestated number of carbon atoms and at least one heteroatom selected fromthe group consisting of O, N, Si, P and S, and wherein the nitrogen,phosphorous and sulfur atoms are optionally oxidized, and the nitrogenheteroatom is optionally be quaternized. The heteroatom(s) O, N, P, Sand Si may be placed at any interior position of the heteroalkyl groupor at the position at which the alkyl group is attached to the remainderof the molecule. Examples include, but are not limited to,—CH₂—CH₂—O—CH₃, —CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃,—CH₂—CH₂, —S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃,—CH₂—CH═N—OCH₃, and —CH═CH—N(CH₃)—CH₃. Up to two heteroatoms may beconsecutive, such as, for example, —CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃.Similarly, the term “heteroalkylene” by itself or as part of anothersubstituent means a divalent radical derived from heteroalkyl, asexemplified, but not limited by, —CH₂—CH₂—S—CH₂—CH₂— and—CH₂—S—CH₂—CH₂—NH—CH₂—. For heteroalkylene groups, heteroatoms can alsooccupy either or both of the chain termini (e.g., alkyleneoxy,alkylenedioxy, alkyleneamino, alkylenediamino, and the like). Stillfurther, for alkylene and heteroalkylene linking groups, no orientationof the linking group is implied by the direction in which the formula ofthe linking group is written. For example, the formula —C(O)₂R′—represents both —C(O)₂R′— and —R′C(O)₂—.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Examples ofcycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic moiety that can be a single ring or multiple rings (preferablyfrom 1 to 3 rings), which are fused together or linked covalently. Theterm “heteroaryl” refers to aryl groups (or rings) that contain from oneto four heteroatoms selected from N, O, and S, wherein the nitrogen andsulfur atoms are optionally oxidized, and the nitrogen atom(s) areoptionally quaternized. A heteroaryl group can be attached to theremainder of the molecule through a heteroatom. Non-limiting examples ofaryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl,4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl,2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl,2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, tetrazolyl, benzo[b]furanyl, benzo[b]thienyl,2,3-dihydrobenzo[1,4]dioxin-6-yl, benzo[1,3]dioxol-5-yl and 6-quinolyl.Substituents for each of the above noted aryl and heteroaryl ringsystems are selected from the group of acceptable substituents describedbelow.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroarylrings as defined above. Thus, the term “arylalkyl” is meant to includethose radicals in which an aryl group is attached to an alkyl group(e.g., benzyl, phenethyl, pyridylmethyl and the like) including thosealkyl groups in which a carbon atom (e.g., a methylene group) has beenreplaced by, for example, an oxygen atom (e.g., phenoxymethyl,2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like).

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “aryl” and“heteroaryl”) includes both substituted and unsubstituted forms of theindicated radical. Preferred substituents for each type of radical areprovided below.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) are generically referred to as “alkyl groupsubstituents,” and they can be one or more of a variety of groupsselected from, but not limited to: —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R′R′″, —OC(O)R′, —C(O)R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂ in a number ranging from zero to (2 m′+1), where m′ is the totalnumber of carbon atoms in such radical. R′, R″, R′″ and R′″ eachpreferably independently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, e.g., aryl substitutedwith 1-3 halogens, substituted or unsubstituted alkyl, alkoxy orthioalkoxy groups, or arylalkyl groups. When a compound of the inventionincludes more than one R group, for example, each of the R groups isindependently selected as are each R′, R″, R″ and R′″ groups when morethan one of these groups is present. When R′ and R″ are attached to thesame nitrogen atom, they can be combined with the nitrogen atom to forma 5-, 6-, or 7-membered ring. For example, —NR′R″ is meant to include,but not be limited to, 1-pyrrolidinyl and 4-morpholinyl. From the abovediscussion of substituents, one of skill in the art will understand thatthe term “alkyl” is meant to include groups including carbon atoms boundto groups other than hydrogen groups, such as haloalkyl (e.g., —CF₃ and—CH₂CF₃) and acyl (e.g., —C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and thelike).

Similar to the substituents described for the alkyl radical,substituents for the aryl and heteroaryl groups are generically referredto as “aryl group substituents.” The substituents are selected from, forexample: halogen, —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′, -halogen,—SiR′R″R′″, —OC(O)R′, —C(O)R′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′,—NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″, —NR—C(NR′R″)═NR′″,—S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and —NO₂, —R′, —N₃,—CH(Ph)₂, fluoro(C₁-C₄)alkoxy, and fluoro(C₁-C₄)alkyl, in a numberranging from zero to the total number of open valences on the aromaticring system; and where R′, R″, R′″ and R′″ are preferably independentlyselected from hydrogen, substituted or unsubstituted alkyl, substitutedor unsubstituted heteroalkyl, substituted or unsubstituted aryl andsubstituted or unsubstituted heteroaryl. When a compound of theinvention includes more than one R group, for example, each of the Rgroups is independently selected as are each R′, R″, R″ and R′″ groupswhen more than one of these groups is present. In the schemes thatfollow, the symbol X represents “R” as described above.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally be replaced with a substituent of the formula-T-C(O)—(CRR′)_(q)—U—, wherein T and U are independently —NR—, —O—,—CRR′— or a single bond, and q is an integer of from 0 to 3.Alternatively, two of the substituents on adjacent atoms of the aryl orheteroaryl ring may optionally be replaced with a substituent of theformula -A-(CH₂)_(r)—B—, wherein A and B are independently —CRR′—, —O—,—NR—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR′— or a single bond, and r is aninteger of from 1 to 4. One of the single bonds of the new ring soformed may optionally be replaced with a double bond. Alternatively, twoof the substituents on adjacent atoms of the aryl or heteroaryl ring mayoptionally be replaced with a substituent of the formula—(CRR′)_(s)—X—(CR″R′″)_(d)—, where s and d are independently integers offrom 0 to 3, and X is O—, —NR′—, —S—, —S(O)—, —S(O)₂—, or —S(O)₂NR′—.The substituents R, R′, R″ and R′″ are preferably independently selectedfrom hydrogen or substituted or unsubstituted (C₁-C₆)alkyl.

As used herein, the term “heteroatom” includes oxygen (O), nitrogen (N),sulfur (S), phosphorus (P) and silicon (Si).

The term “amino” or “amine group” refers to the group —NR′R″ (orN⁺RR′R″) where R, R′ and R″ are independently selected from the groupconsisting of hydrogen, alkyl, substituted alkyl, aryl, substitutedaryl, aryl alkyl, substituted aryl alkyl, heteroaryl, and substitutedheteroaryl. A substituted amine being an amine group wherein R′ or R″ isother than hydrogen. In a primary amino group, both R′ and R″ arehydrogen, whereas in a secondary amino group, either, but not both, R′or R″ is hydrogen. In addition, the terms “amine” and “amino” caninclude protonated and quaternized versions of nitrogen, comprising thegroup —N⁺RR′R″ and its biologically compatible anionic counterions.

The term “aqueous solution” as used herein refers to a solution that ispredominantly water and retains the solution characteristics of water.Where the aqueous solution contains solvents in addition to water, wateris typically the predominant solvent.

The term “buffer” as used herein refers to a system that acts tominimize the change in acidity or basicity of the solution againstaddition or depletion of chemical substances.

The term “carbonyl” as used herein refers to the functional group—(C═O)—. However, it will be appreciated that this group may be replacedwith other well-known groups that have similar electronic and/or stericcharacter, such as thiocarbonyl (—(C═S)—); sulfinyl (—S(O)—); sulfonyl(—SO₂)—), phosphonyl (—PO₂—).

The term “Carboxyalkyl” as used herein refers to a group having thegeneral formula —(CH₂)_(n)COOH wherein n is 1-18.

The term “carrier molecule” as used herein refers to a fluorogenic,fluorescent or colorimetric compound of the present invention that iscovalently bonded to a biological or a non-biological component. Suchcomponents include, but are not limited to, an amino acid, a peptide, aprotein, a polysaccharide, a nucleoside, a nucleotide, anoligonucleotide, a nucleic acid, a hapten, a psoralen, a drug, ahormone, a lipid, a lipid assembly, a synthetic polymer, a polymericmicroparticle, a biological cell, a virus and combinations thereof.

The term “detectable response” as used herein refers to a change in oran occurrence of, a signal that is directly or indirectly detectableeither by observation or by instrumentation. Typically, the detectableresponse is an optical response resulting in a change in the wavelengthdistribution patterns or intensity of absorbance or fluorescence or achange in light scatter, fluorescence lifetime, fluorescencepolarization, or a combination of the above parameters.

The term “enzymatic peroxide producing system” used herein refers to asystem comprising an enzyme and an appropriate substrate wherein atleast one of the resulting products is peroxide.

The term “enzyme” as used herein refers to a peptide with catalyticactivity.

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl,” aremeant to include monohaloalkyl and polyhaloalkyl. For example, the term“halo(C₁-C₄)alkyl” is mean to include, but not be limited to,trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, andthe like.

The term “kit” as used refers to a packaged set of related components,typically one or more compounds or compositions.

The term “Linker” or “L”, as used herein, refers to a single covalentbond or a series of stable covalent bonds incorporating 1-20 nonhydrogenatoms selected from the group consisting of C, N, O, S and P thatcovalently attach the fluorogenic or fluorescent compounds to anothermoiety such as a chemically reactive group or a biological andnon-biological component. Exemplary linking members include a moietythat includes —C(O)NH—, —C(O)O—, —NH—, —S—, —O—, and the like. A“cleavable linker” is a linker that has one or more cleavable groupsthat may be broken by the result of a reaction or condition. The term“cleavable group” refers to a moiety that allows for release of aportion, e.g., a fluorogenic or fluorescent moiety, of a conjugate fromthe remainder of the conjugate by cleaving a bond linking the releasedmoiety to the remainder of the conjugate. Such cleavage is eitherchemical in nature, or enzymatically mediated. Exemplary enzymaticallycleavable groups include natural amino acids or peptide sequences thatend with a natural amino acid.

In addition to enzymatically cleavable groups, it is within the scope ofthe present invention to include one or more sites that are cleaved bythe action of an agent other than an enzyme. Exemplary non-enzymaticcleavage agents include, but are not limited to, acids, bases, light(e.g., nitrobenzyl derivatives, phenacyl groups, benzoin esters), andheat. Many cleaveable groups are known in the art. See, for example,Jung et al., Biochem. Biophys. Acta, 761: 152-162 (1983); Joshi et al.,J. Biol. Chem., 265: 14518-14525 (1990); Zarling et al., J. Immunol.,124: 913-920 (1980); Bouizar et al., Eur. J. Biochem., 155: 141-147(1986); Park et al., J. Biol. Chem., 261: 205-210 (1986); Browning etal., J. Immunol., 143: 1859-1867 (1989). Moreover a broad range ofcleavable, bifunctional (both homo- and hetero-bifunctional) spacer armsare commercially available.

An exemplary cleavable group, an ester, is cleavable group that may becleaved by a reagent, e.g. sodium hydroxide, resulting in acarboxylate-containing fragment and a hydroxyl-containing product.

The linker can be used to attach the compound to another component of aconjugate, such as a targeting moiety (e.g., antibody, ligand,non-covalent protein-binding group, etc.), an analyte, a biomolecule, adrug and the like.

As used herein “peroxidase” refers to an enzyme that catalyzes theoxidation of a molecule by a peroxide. This includes all enzymes thatcontain peroxidase activity, including, but not limited to,cyclooxygenase, horseradish peroxidase and myeloperoxidase.

As used herein “peroxide” refers to a molecule that includes the —O—O—moiety.

The term “protecting group,” as used herein refers to a portion of asubstrate that is substantially stable under a particular reactioncondition, but which is cleaved from the substrate under a differentreaction condition. A protecting group can also be selected such that itparticipates in the direct oxidation of the aromatic ring component ofthe compounds of the invention. For examples of useful protectinggroups, see, for example, Greene et al., PROTECTIVE GROUPS IN ORGANICSYNTHESIS, John Wiley & Sons, New York, 1991.

The term “reactive group” as used herein refers to a group that iscapable of reacting with another chemical group to form a covalent bond,i.e. is covalently reactive under suitable reaction conditions, andgenerally represents a point of attachment for another substance. Thereactive group is a moiety, such as carboxylic acid or succinimidylester, on the compounds of the present invention that is capable ofchemically reacting with a functional group on a different compound toform a covalent linkage resulting in a fluorescent or fluorogeniclabeled component. Reactive groups generally include nucleophiles,electrophiles and photoactivatable groups.

Exemplary reactive groups include, but not limited to, olefins,acetylenes, alcohols, phenols, ethers, oxides, halides, aldehydes,ketones, carboxylic acids, esters, amides, cyanates, isocyanates,thiocyanates, isothiocyanates, amines, hydrazines, hydrazones,hydrazides, diazo, diazonium, nitro, nitriles, mercaptans, sulfides,disulfides, sulfoxides, sulfones, sulfonic acids, sulfinic acids,acetals, ketals, anhydrides, sulfates, sulfenic acids isonitriles,amidines, imides, imidates, nitrones, hydroxylamines, oximes, hydroxamicacids thiohydroxamic acids, allenes, ortho esters, sulfites, enamines,ynamines, ureas, pseudoureas, semicarbazides, carbodiimides, carbamates,imines, azides, azo compounds, azoxy compounds, and nitroso compounds.Reactive functional groups also include those used to preparebioconjugates, e.g., N-hydroxysuccinimide esters, maleimides and thelike. Methods to prepare each of these functional groups are well knownin the art and their application to or modification for a particularpurpose is within the ability of one of skill in the art (see, forexample, Sandler and Karo, eds. ORGANIC FUNCTIONAL GROUP PREPARATIONS,Academic Press, San Diego, 1989).

The term “salt thereof,” as used herein includes salts of the agents ofthe invention and their conjugates, which are preferably prepared withrelatively nontoxic acids or bases, depending on the particularsubstituents found on the compounds described herein. When compounds ofthe present invention contain relatively acidic functionalities, baseaddition salts can be obtained by contacting the neutral form of suchcompounds with a sufficient amount of the desired base, either neat orin a suitable inert solvent. Examples of base addition salts includesodium, potassium, calcium, ammonium, organic amino, or magnesium, or asimilar salt. When compounds of the present invention contain relativelybasic functionalities, acid addition salts can be obtained by contactingthe neutral form of such compounds with a sufficient amount of thedesired acid, either neat or in a suitable inert solvent. Examples ofaddition salts include those derived from inorganic acids likehydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic,phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived from relatively nontoxic organic acids likeacetic, propionic, isobutyric, maleic, malonic, benzoic, succinic,suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic,p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Alsoincluded are salts of amino acids such as arginate and the like, andsalts of organic acids like glucuronic or galactunoric acids and thelike (see, for example, Berge et al., “Pharmaceutical Salts”, Journal ofPharmaceutical Science, 1977, 66, 1-19). Certain specific compounds ofthe present invention contain both basic and acidic functionalities thatallow the compounds to be converted into either base or acid additionsalts.

The term “sample” as used herein refers to any material that may containa peroxide. The sample may also include diluents, buffers, detergents,and contaminating species, debris and the like that are found mixed withthe target. Illustrative examples include urine, sera, blood plasma,total blood, saliva, tear fluid, cerebrospinal fluid, secretory fluidsfrom nipples and the like. Also included are solid, gel or solsubstances such as mucus, body tissues, cells and the like suspended ordissolved in liquid materials such as buffers, extractants, solvents andthe like. Typically, the sample is a live cell, a biological fluid thatcomprises endogenous host cell proteins, nucleic acid polymers,nucleotides, oligonucleotides, peptides and buffer solutions. The samplemay be in an aqueous solution, a viable cell culture or immobilized on asolid or semi solid surface such as a polyacrylamide gel, membrane blotor on a microarray.

The term “solid support,” as used herein, refers to a material that issubstantially insoluble in a selected solvent system, or which can bereadily separated (e.g., by precipitation) from a selected solventsystem in which it is soluble. Solid supports useful in practicing thepresent invention can include groups that are activated or capable ofactivation to allow selected species to be bound to the solid support.Solid supports may be present in a variety of forms, including a chip,wafer or well, onto which an individual, or more than one compound, ofthe invention is bound such as a polymeric bead or particle.

The term “sulfoalkyl,” as used herein refers to a group having thegeneral formula —(CH₂)_(n)SO₃ wherein n is 1-18.

The term “targeting group” as used herein refers to a moiety that is:(1) able to actively direct the entity to which it is attached (e.g., afluorogenic moiety) to a target region, e.g., a cell; or (2) ispreferentially passively absorbed by or entrained within a targetregion. The targeting group can be a small molecule, which is intendedto include both non-peptides and peptides. The targeting group can alsobe a macromolecule, which includes, but is not limited to, saccharides,lectins, receptors, ligand for receptors, proteins such as BSA,antibodies, poly(ethers), dendrimers, poly(amino acids) and so forth.

The Compounds

The present invention provides both fluorogenic and fluorescentcompounds based on a ring system according to Formula I:

in which the symbol R^(a) represents H, substituted or unsubstitutedalkyl, substituted or unsubstituted aryl, O, and C(X^(a))R^(a1), inwhich X^(a) represents O, S or NH and R^(a1) is a member selected fromsubstituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted acyl, OR²² and NR^(a3)R^(a4). The symbolsR^(a2), R^(a3) and R^(a4) independently represent moieties such ashydrogen, substituted alkyl, unsubstituted alkyl, substitutedheteroalkyl, unsubstituted heteroalkyl, substituted heterocycloalkyl,unsubstituted heterocycloalkyl, substituted carboxyalkyl, unsubstitutedcarboxyalkyl, substituted sulfoalkyl, unsubstituted sulfoalkyl,substituted acyl, unsubstituted acyl, substituted haloalkyl,unsubstituted haloalkyl, substituted alkoxy, unsubstituted alkoxy, areactive group, substituted reactive group, carrier molecule,substituted carrier molecule, solid support or substituted solidsupport. Alternatively, a member independently selected from R^(d1) incombination with R^(d2); R^(d1) in combination with R^(c); R^(d1) incombination with R^(e); R^(d1) in combination with R^(f); and R^(d2) incombination with R^(i) together with the atoms to which they are joined,form a ring which is a 5-, 6- or 7-membered heterocycloalkyl, asubstituted 5-, 6- or 7-membered heterocycloalkyl, a 5-, 6- or7-membered heteroaryl, or a substituted 5-, 6- or 7-membered heteroaryl.

The index “n” represents either 0 or 1.

R^(b), R^(c), R^(e), R^(f), R^(h) and R^(i) are members that areindependently selected from the genus of aryl substituents, includingspecies such as H, OH, sulfo, nitro, carboxyl, carboxylate esters,halogen, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl (e.g., alkoxy, alkylthio, aminoalkyl, etc.),substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted heterocycloalkyl, a reactivegroup, substituted reactive group, a carrier molecule, substitutedcarrier molecule, a solid support or substituted solid support.Alternatively, a member independently selected from R^(b) in combinationwith R^(c); and R^(h) in combination with R^(i) together with the atomsto which they are joined, form a ring which is a 5-, 6- or 7-memberedcycloalkyl, a substituted 5-, 6- or 7-membered cycloalkyl, a 5-, 6- or7-membered heterocycloalkyl, a substituted 5-, 6- or 7-memberedheterocycloalkyl, a 5-, 6- or 7-membered aryl, a substituted 5-, 6- or7-membered aryl, a 5-, 6- or 7-membered heteroaryl, or a substituted 5-,6- or 7-membered heteroaryl. At least one of R^(b), R^(c), R^(e), R^(f),R^(h) and R^(i) is fluorine.

The symbol R^(d) represents OR^(d1) or NR^(d1)R^(d2). The identities ofR^(d1) and R^(d2) are the same as those set forth for R^(a1); R^(a3) andR^(a4). R^(g) represents OR^(g1), NR^(g1) and R^(g2) or (═O). Theidentities of R^(g1) and R^(g2) are the same as those set forth abovefor R^(d1) and R^(d2).

The dashed lines in the formula represent double bonds that are eitherpresent or absent as required to satisfy the rules of valency. Thus, theformula above includes the following exemplary substructures:

In an exemplary embodiment, the invention provides a compound accordingto Formula I that has the structure:

in which the symbols A and E independently represent a member selectedfrom OR⁸ and NR⁹R¹⁰. R⁹ is hydrogen, substituted alkyl, unsubstitutedalkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substitutedheterocycloalkyl, unsubstituted heterocycloalkyl, substitutedcarboxyalkyl, unsubstituted carboxyalkyl, substituted sulfoalkyl,unsubstituted sulfoalkyl, substituted acyl, unsubstituted acyl,substituted haloalkyl, unsubstituted haloalkyl, substituted alkoxy,unsubstituted alkoxy, a reactive group, substituted reactive group,carrier molecule, substituted carrier molecule, solid support orsubstituted solid support. The groups R⁹ and R¹⁰ are independentlyhydrogen, substituted alkyl, unsubstituted alkyl, substitutedheteroalkyl, unsubstituted heteroalkyl, substituted heterocycloalkyl,unsubstituted heterocycloalkyl, substituted carboxyalkyl, unsubstitutedcarboxyalkyl, substituted sulfoalkyl, unsubstituted sulfoalkyl,substituted acyl, unsubstituted acyl, substituted haloalkyl,unsubstituted haloalkyl, substituted alkoxy, unsubstituted alkoxy, areactive group, substituted reactive group, carrier molecule,substituted carrier molecule, solid support or substituted solidsupport.

Selected substituents, together with the ring system atoms to which theyare attached, are optionally joined to form a ring which is a 5-, 6- or7-membered heterocycloalkyl, a substituted 5-, 6- or 7-memberedheterocycloalkyl, a 5-, 6- or 7-membered heteroaryl, or a substituted5-, 6- or 7-membered heteroaryl. Exemplary substituents that can becyclized in this manner include R⁹ in combination with R¹⁰; R⁹ incombination with R³; R⁹ in combination with R⁶; R¹⁰ in combination withR⁴; and R¹⁰ in combination with R⁵. X is either oxygen or sulfur.

R¹ is a substituted alkyl, unsubstituted alkyl, substituted heteroalkyl,unsubstituted heteroalkyl, substituted aryl, unsubstituted aryl,substituted heteroaryl, unsubstituted heteroaryl, substitutedheterocycloalkyl, unsubstituted heterocycloalkyl, OR⁸ or NR⁹R¹⁰. X¹ iseither oxygen or sulfur.

The symbols R², R³, R⁴, R⁵, R⁶ and R⁷ independently represent hydrogen,halogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy,unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio,substituted aryl, unsubstituted aryl, substituted heteroaryl,unsubstituted heteroaryl, sulfo, nitro, carboxyl, hydroxyl, a reactivegroup, substituted reactive group, a carrier molecule, substitutedcarrier molecule, a solid support or substituted solid support. In anexemplary embodiment, at least one of R², R³, R⁴, R⁵, R⁶ and R⁷ isfluorine.

In one embodiment, the present compounds are according to the formula:

wherein R¹ is a substituted alkyl, unsubstituted alkyl, substitutedheteroalkyl, unsubstituted heteroalkyl, substituted aryl, unsubstitutedaryl, substituted heteroaryl, unsubstituted heteroaryl, substitutedheterocycloalkyl, unsubstituted heterocycloalkyl, OR⁸ or NR⁹R¹⁰. Theidentities of R², R³, R⁴, R⁵, R⁶, R⁷, X, A and E are the same as thoseset forth above.

In an exemplary embodiment R¹ is an alkyl, typically methyl, A and E areboth OR⁸, wherein R⁸ is typically an alkyl or hydrogen and wherein atleast one of R², R³, R⁴, R⁵, R⁶ or R⁷ is fluorine.

Thus is one aspect, the present compounds are according to the formula:

In one aspect, at least one of R³, R⁴, R⁵, and R⁶ is fluorine. Inanother aspect R³ and R⁶ are each fluorine. In a further aspect, R³ andR⁶ are each fluorine and R², R⁴, R⁵ and R⁷ are hydrogen, substitutedalkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy,substituted alkylthio, unsubstituted alkylthio, substituted aryl,unsubstituted aryl, substituted heteroaryl, or unsubstituted heteroaryl.Typically R², R⁴, R⁵ and R⁷ are each hydrogen.

In another aspect, R⁴ and R⁵ are each fluorine wherein R², R³, R⁶ and R⁷are hydrogen, substituted alkyl, unsubstituted alkyl, substitutedalkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstitutedalkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl,or unsubstituted heteroaryl. In a further aspect, R², R³, R⁶ and R⁷ areeach hydrogen.

In yet another aspect, R³, R⁴, R⁵ and R⁶ are each fluorine wherein R²and R⁷ are hydrogen, substituted alkyl, unsubstituted alkyl, substitutedalkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstitutedalkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl,or unsubstituted heteroaryl.

In another exemplary embodiment, A and E are each NR⁹R¹⁰ and R⁹ and R¹⁰are independently hydrogen, substituted alkyl or unsubstituted alkyl.Typically R⁹ and R¹⁰ are each hydrogen.

Exemplary compounds according to Formula II(b) of the invention includethose having the formulae:

In certain embodiments, the compounds of the present invention have theformula:

In this instance R¹ is a substituted alkyl, unsubstituted alkyl,substituted heteroalkyl, unsubstituted heteroalkyl, substituted aryl,unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl,substituted heterocycloalkyl, unsubstituted heterocycloalkyl, OR⁸ orNR⁹R¹⁰. The identities of R², R³, R⁴, R⁵, R⁶, R⁷, X, A and E are thesame as those set forth above.

In another exemplary embodiment, the invention provides compoundsaccording to Formula III:

wherein A is OR⁸ or NR⁹R¹⁰, Z is either oxygen (O) or a quaternizedamine, N⁺R⁹R¹⁰. At least one of R², R³, R⁴, R⁵, R⁶ and R⁷ is fluorine.The identities of R², R³, R⁴, R⁵, R⁶ and R⁷ are the same as those setabove.

The compounds according to Formula (III) are inherently fluorescent andare generally referred to as oxazine dyes or thiazine dyes. They can beused with the many methods of the present invention, including asspectrally matched controls for the fluorogenic compounds according toformula II. It is understood that the fluorogenic compounds according toFormula II (hydroxyl derivative) are converted into correspondingFormula III (ketone derivative) compounds after reaction with peroxide.

In an exemplary embodiment A is OR⁸ and Z is O, wherein R⁸ is typicallyan alkyl or hydrogen and wherein at least one of R², R³, R⁴, R⁵, R⁶ orR⁷ is fluorine.

Thus is one aspect, the present compounds are according to the formula:

In one aspect, at least one of R³, R⁴, R⁵, and R⁶ is fluorine. Inanother aspect R³ and R⁶ are each fluorine. In a further aspect, R³ andR⁶ are each fluorine and R², R⁴, R⁵ and R⁷ are hydrogen, substitutedalkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy,substituted alkylthio, unsubstituted alkylthio, substituted aryl,unsubstituted aryl, substituted heteroaryl, or unsubstituted heteroaryl.Typically R², R⁴, R⁵ and R⁷ are each hydrogen.

In another aspect, R⁴ and R⁵ are each fluorine wherein R², R³, R⁶ and R⁷are hydrogen, substituted alkyl, unsubstituted alkyl, substitutedalkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstitutedalkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl,or unsubstituted heteroaryl. In a further aspect, R², R³, R⁶ and R⁷ areeach hydrogen.

In yet another aspect, R³, R⁴, R⁵ and R⁶ are each fluorine wherein R²and R⁷ are hydrogen, substituted alkyl, unsubstituted alkyl, substitutedalkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstitutedalkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl,or unsubstituted heteroaryl.

In another exemplary embodiment, A is NR⁹R¹⁰ and Z is N⁺R⁹R¹⁰ and R⁹ andR¹⁰ are independently hydrogen, substituted alkyl or unsubstitutedalkyl. Typically R⁹ and R¹⁰ are each hydrogen.

In an exemplary embodiment, at least one of R², R³, R⁴, R⁵, R⁶ and R⁷,which is not a fluoro, is a reactive group, a carrier molecule or solidsupport. The atoms and groups represented by symbols in Formula IIIother than those discussed explicitly above are substantially identicalto those described for the compounds of Formula II.

In still a further exemplary embodiment, the compounds of the inventionhave a structure according to Formula IV:

in which the atoms and groups represented by the symbols in the formulaare substantially identical to those described for the correspondingsymbols of Formula III, provided that at least one of the R groups isfluorine. Compounds according to Formula (IV) are fluorogenic and asgenerally referred to as resazurin compounds.

In an exemplary embodiment A is OR⁸ and Z is O, wherein R⁸ is typicallyan alkyl or hydrogen and wherein at least one of R², R³, R⁴, R⁵, R⁶ orR⁷ is fluorine.

In one aspect, at least one of R³, R⁴, R⁵, and R⁶ is fluorine. Inanother aspect R³ and R⁶ are each fluorine. In a further aspect, R³ andR⁶ are each fluorine and R², R⁴, R⁵ and R⁷ are hydrogen, substitutedalkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy,substituted alkylthio, unsubstituted alkylthio, substituted aryl,unsubstituted aryl, substituted heteroaryl, or unsubstituted heteroaryl.Typically R², R⁴, R⁵ and R⁷ are each hydrogen.

In another aspect, R⁴ and R⁵ are each fluorine wherein R², R³, R⁶ and R⁷are hydrogen, substituted alkyl, unsubstituted alkyl, substitutedalkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstitutedalkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl,or unsubstituted heteroaryl. In a further aspect, R², R³, R⁶ and R⁷ areeach hydrogen.

In yet another aspect, R³, R⁴, R⁵ and R⁶ are each fluorine wherein R²and R⁷ are hydrogen, substituted alkyl, unsubstituted alkyl, substitutedalkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstitutedalkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl,or unsubstituted heteroaryl.

In another exemplary embodiment, A is NR⁹R¹⁰ and Z is N⁺R⁹R¹⁰ and R⁹ andR¹⁰ are independently hydrogen, substituted alkyl or unsubstitutedalkyl. Typically R⁹ and R¹⁰ are each hydrogen.

An exemplary compound according to Formula IV is compound 32:

Synthesis

In an exemplary route, the compounds of the invention are preparedstarting with a fluorinated resorcinol, which is nitrosated andcondensed with another resorcinol. Prior to the present invention, itwas not recognized that a nitrosofluororesorcinol could be prepared andcondensed with a resorcinol partner to produce the fused ring systemdescribed herein. Thus, it is an aspect of the present invention toprepare fluorogenic and fluorescent compounds according to Formulae I-IVby a route according to Scheme 1. In Scheme I, the synthesis begins withthe nitrosation of a selected resorcinol. The precursor resorcinol maybe substituted at one or more of the 2-, 5-, or 6-position(s). In anexemplary synthetic route, the resulting 4-nitroso-resorcinol (a) iscondensed in warm acid with a resorcinol (b) that may be substituted atthe 2-, 4-, and/or 5-positions, but preferably not at the 6-position.The resulting resorufin (c) is reduced to the dihydro form (d). Thesecondary amine moiety in d is optionally acylated, affording e. Atleast one of Y^(a), Y^(a′), Y^(b), Y^(b′), Y^(c), Y^(c′) is fluorine.

The reduced derivatives of fluorinated resorufin dyes are prepared bychemical reduction of the ketone moiety portion with stannous chloridein organic solvents. These dihydroxy-fluorinated compounds can serve assubstrates for enzymes that take up electrons, or in the detection ofchemical oxidizing agents, reactive oxygen species or nitric oxides.

Several other moieties are optionally present during these syntheticsteps; they may either be protected with a protecting group or resistantto the steps required for synthesis of the fluorinated compound.Exemplary moieties include alkyl, carboxyalkyl, chloro, bromo, iodo,alkoxy and hydroxy. Hydroxy moieties may also be formed during cleavageof alkoxy groups present in the starting material.

The dihydroxyphenoxazine and phenoxazin-3-one versions of the dyes ofthe invention and their conjugates are generally freely interconvertibleby chemical oxidation or reduction. Reagents useful for this reductioninclude borohydrides, aluminum hydrides, hydrogen in the presence of ahydrogenation catalyst, and dithionites. Choice of the reducing agentmay depend on the ease of reduction of other reducible groups in themolecule.

A wide variety of oxidizing agents mediate the oxidation of thedihydroxyphenoxazine, including molecular oxygen in the presence orabsence of a catalyst, peroxide, nitric oxide, peroxynitrite,dichromate, triphenylcarbenium and chloranil. The dihydroxyphenoxazinesare also oxidized by enzymatic action, including the action ofhorseradish peroxidase in combination with peroxides (Example 33; FIG.1), by cyclooxygenase (Example 34; FIG. 2), hemoglobin (Example 35; FIG.3) or solely by nitric oxide. This oxidation may occur in a cell, or ina cell-free solution.

Post-condensation modifications of the fluorinated keto- anddihydroxy-phenoxazines are similar to known methods of modifying theirnon-fluorinated analogues.

The compounds of the invention also exist in other enzyme substrateformats. For example, the fluorinated compounds can be derivatized at aphenolic hydroxyl with phosphate (to give phosphatase substrates),carboxylic acids (to give esterase substrates), alkylation with alkylgroups (to give dealkylase substrates) and carbohydrates to yieldglycosidase substrates. Moreover, the invention provides compounds inwhich the ring system, or a substituent thereon, includes a lipophilicmoiety, such as a long chain fatty acid or alcohol moiety, or aphospholipids.

Reactive Groups, Carrier Molecules and Solid Supports

The present compounds, in certain embodiments, are chemically reactivewherein the compounds comprise a reactive group. In a furtherembodiment, the compounds comprise a carrier molecule or solid support.These substituents, reactive groups, carrier molecules, and solidsupports, comprise a linker that is used to covalently attach thesubstituents to any of the moieties of the present compounds. The solidsupport, carrier molecule or reactive group may be directly attached(where linker is a single bond) to the moieties or attached through aseries of stable bonds, as disclosed above.

Any combination of linkers may be used to attach the carrier molecule,solid support or reactive group and the present compounds together. Thelinker may also be substituted to alter the physical properties of thereporter moiety or chelating moiety, such as spectral properties of thedye.

The linker typically incorporates 1-30 nonhydrogen atoms selected fromthe group consisting of C, N, O, S and P. The linker may be anycombination of stable chemical bonds, optionally including, single,double, triple or aromatic carbon-carbon bonds, as well ascarbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygen bonds,sulfur-sulfur bonds, carbon-sulfur bonds, phosphorus-oxygen bonds,phosphorus-nitrogen bonds, and nitrogen-platinum bonds. Typically thelinker incorporates less than 15 nonhydrogen atoms and are composed ofany combination of ether, thioether, thiourea, amine, ester,carboxamide, sulfonamide, hydrazide bonds and aromatic or heteroaromaticbonds. Typically the linker is a combination of single carbon-carbonbonds and carboxamide, sulfonamide or thioether bonds. The bonds of thelinker typically result in the following moieties that can be found inthe linker: ether, thioether, carboxamide, thiourea, sulfonamide, urea,urethane, hydrazine, alkyl, aryl, heteroaryl, alkoxy, cycloalkyl andamine moieties. Examples of a linker include substituted orunsubstituted polymethylene, arylene, alkylarylene, arylenealkyl, andarylthio.

In one embodiment, the linker contains 1-6 carbon atoms; in another, thelinker comprises a thioether linkage. Exemplary linking members includea moiety that includes —C(O)NH—, —C(O)O—, —NH—, —S—, —O—, and the like.In another embodiment, the linker is or incorporates the formula—(CH₂)_(d)(CONH(CH₂)_(e))_(z)— or where d is an integer from 0-5, e isan integer from 1-5 and z is 0 or 1. In a further embodiment, the linkeris or incorporates the formula —O—(CH₂)—. In yet another embodiment, thelinker is or incorporates a phenylene or a 2-carboxy-substitutedphenylene.

An important feature of the linker is to provide an adequate spacebetween the carrier molecule, reactive group or solid support and thedye so as to prevent steric hinderance. Therefore, the linker of thepresent compound is important for (1) attaching the carrier molecule,reactive group or solid support to the compound, (2) providing anadequate space between the carrier molecule, reactive group or solidsupport and the compound so as not to sterically hinder the action ofthe compound and (3) for altering the physical properties of the presentcompounds.

In another exemplary embodiment of the invention, the present compoundsare chemically reactive, and are substituted by at least one reactivegroup. The reactive group functions as the site of attachment foranother moiety, such as a carrier molecule or a solid support, whereinthe reactive group chemically reacts with an appropriate reactive orfunctional group on the carrier molecule or solid support.

In an exemplary embodiment, the compounds of the invention furthercomprise a reactive group which is a member selected from an acrylamide,an activated ester of a carboxylic acid, a carboxylic ester, an acylazide, an acyl nitrile, an aldehyde, an alkyl halide, an anhydride, ananiline, an amine, an aryl halide, an azide, an aziridine, a boronate, adiazoalkane, a haloacetamide, a haloalkyl, a halotriazine, a hydrazine,an imido ester, an isocyanate, an isothiocyanate, a maleimide, aphosphoramidite, a photoactivatable group, a reactive platinum complex,a silyl halide, a sulfonyl halide, and a thiol. In a particularembodiment the reactive group is selected from the group consisting ofcarboxylic acid, succinimidyl ester of a carboxylic acid, hydrazide,amine and a maleimide. In exemplary embodiment, at least one memberselected from R^(b), R^(c), R^(d1), R^(d2), R^(e), R^(f), R^(h), R², R³,R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, or R¹⁰ comprises a reactive group. Preferably,at least one of R^(b), R^(c), R^(e), R^(f), R^(h), R^(i), R², R³, R⁴,R⁵, R⁶, or R⁷ comprises a reactive group or is attached to a reactivegroup. Alternatively, if the present compound comprises a carriermolecule or solid support a reactive group may be covalently attachedindependently to those substituents, allowing for further conjugation toa another dye, carrier molecule or solid support.

In one aspect, the compound comprises at least one reactive group thatselectively reacts with an amine group. This amine-reactive group isselected from the group consisting of succinimidyl ester, sulfonylhalide, tetrafluorophenyl ester and iosothiocyanates. Thus, in oneaspect, the present compounds form a covalent bond with anamine-containing molecule in a sample. In another aspect, the compoundcomprises at least one reactive group that selectively reacts with athiol group. This thiol-reactive group is selected from the groupconsisting of maleimide, haloalkyl and haloacetamide (including anyreactive groups disclosed in U.S. Pat. Nos. 5,362,628; 5,352,803 and5,573,904).

The pro-reactive groups are synthesized during the formation of themonomer moieties and carrier molecule and solid support containingcompounds to provide chemically reactive compounds. In this way,compounds incorporating a reactive group can be covalently attached to awide variety of carrier molecules or solid supports that contain or aremodified to contain functional groups with suitable reactivity,resulting in chemical attachment of the components. In an exemplaryembodiment, the reactive group of the compounds of the invention and thefunctional group of the carrier molecule or solid support compriseelectrophiles and nucleophiles that can generate a covalent linkagebetween them. Alternatively, the reactive group comprises aphotoactivatable group, which becomes chemically reactive only afterillumination with light of an appropriate wavelength. Typically, theconjugation reaction between the reactive group and the carrier moleculeor solid support results in one or more atoms of the reactive groupbeing incorporated into a new linkage attaching the present compound ofthe invention to the carrier molecule or solid support. Selectedexamples of functional groups and linkages are shown in Table 1, wherethe reaction of an electrophilic group and a nucleophilic group yields acovalent linkage.

TABLE 1 Examples of some routes to useful covalent linkages ResultingCovalent Electrophilic Group Nucleophilic Group Linkage activatedesters* amines/anilines carboxamides acrylamides thiols thioethers acylazides** amines/anilines carboxamides acyl halides amines/anilinescarboxamides acyl halides alcohols/phenols esters acyl nitrilesalcohols/phenols esters acyl nitriles amines/anilines carboxamidesaldehydes amines/anilines imines aldehydes or ketones hydrazineshydrazones aldehydes or ketones hydroxylamines oximes alkyl halidesamines/anilines alkyl amines alkyl halides carboxylic acids esters alkylhalides thiols thioethers alkyl halides alcohols/phenols ethers alkylsulfonates thiols thioethers alkyl sulfonates carboxylic acids estersalkyl sulfonates alcohols/phenols ethers anhydrides alcohols/phenolsesters anhydrides amines/anilines carboxamides aryl halides thiolsthiophenols aryl halides amines aryl amines aziridines thiols thioethersboronates glycols boronate esters carbodiimides carboxylic acidsN-acylureas or anhydrides diazoalkanes carboxylic acids esters epoxidesthiols thioethers haloacetamides thiols thioethers haloplatinate aminoplatinum complex haloplatinate heterocycle platinum complexhaloplatinate thiol platinum complex halotriazines amines/anilinesaminotriazines halotriazines alcohols/phenols triazinyl ethershalotriazines thiols triazinyl thioethers imido esters amines/anilinesamidines isocyanates amines/anilines ureas isocyanates alcohols/phenolsurethanes isothiocyanates amines/anilines thioureas maleimides thiolsthioethers phosphoramidites alcohols phosphite esters silyl halidesalcohols silyl ethers sulfonate esters amines/anilines alkyl aminessulfonate esters thiols thioethers sulfonate esters carboxylic acidsesters sulfonate esters alcohols ethers sulfonyl halides amines/anilinessulfonamides sulfonyl halides phenols/alcohols sulfonate esters*Activated esters, as understood in the art, generally have the formula—COΩ, where Ω is a good leaving group (e.g., succinimidyloxy (—OC₄H₄O₂)sulfosuccinimidyloxy (—OC₄H₃O₂—SO₃H), -1-oxybenzotriazolyl (—OC₆H₄N₃);or an aryloxy group or aryloxy substituted one or more times by electronwithdrawing substituents such as nitro, fluoro, chloro, cyano, ortrifluoromethyl, or combinations thereof, used to form activated arylesters; or a carboxylic acid activated by a carbodiimide to form ananhydride or mixed anhydride —OCOR^(a) or —OCNR^(a)NHR^(b), where R^(a)and R^(b), which may be the same or different, are C₁-C₆ alkyl, C₁-C₆perfluoroalkyl, or C₁-C₆ alkoxy; or cyclohexyl, 3-dimethylaminopropyl,or N-morpholinoethyl). **Acyl azides can also rearrange to isocyanates

Choice of the reactive group used to attach the compound of theinvention to the substance to be conjugated typically depends on thereactive or functional group on the substance to be conjugated and thetype or length of covalent linkage desired. The types of functionalgroups typically present on the organic or inorganic substances(biomolecule or non-biomolecule) include, but are not limited to,amines, amides, thiols, alcohols, phenols, aldehydes, ketones,phosphates, imidazoles, hydrazines, hydroxylamines, disubstitutedamines, halides, epoxides, silyl halides, carboxylate esters, sulfonateesters, purines, pyrimidines, carboxylic acids, olefinic bonds, or acombination of these groups. A single type of reactive site may beavailable on the substance (typical for polysaccharides or silica), or avariety of sites may occur (e.g., amines, thiols, alcohols, phenols), asis typical for proteins.

Typically, the reactive group will react with an amine, a thiol, analcohol, an aldehyde, a ketone, or with silica. Preferably, reactivegroups react with an amine or a thiol functional group, or with silica.In one embodiment, the reactive group is an acrylamide, an activatedester of a carboxylic acid, an acyl azide, an acyl nitrile, an aldehyde,an alkyl halide, a silyl halide, an anhydride, an aniline, an arylhalide, an azide, an aziridine, a boronate, a diazoalkane, ahaloacetamide, a halotriazine, a hydrazine (including hydrazides), animido ester, an isocyanate, an isothiocyanate, a maleimide, aphosphoramidite, a reactive platinum complex, a sulfonyl halide, or athiol group. By “reactive platinum complex” is particularly meantchemically reactive platinum complexes such as described in U.S. Pat.No. 5,714,327.

Where the reactive group is an activated ester of a carboxylic acid,such as a succinimidyl ester of a carboxylic acid, a sulfonyl halide, atetrafluorophenyl ester or an isothiocyanates, the resulting compound isparticularly useful for preparing conjugates of carrier molecules suchas proteins, nucleotides, oligonucleotides, or haptens. Where thereactive group is a maleimide, haloalkyl or haloacetamide (including anyreactive groups disclosed in U.S. Pat. Nos. 5,362,628; 5,352,803 and5,573,904 (supra)) the resulting compound is particularly useful forconjugation to thiol-containing substances. Where the reactive group isa hydrazide, the resulting compound is particularly useful forconjugation to periodate-oxidized carbohydrates and glycoproteins, andin addition is an aldehyde-fixable polar tracer for cell microinjection.Where the reactive group is a silyl halide, the resulting compound isparticularly useful for conjugation to silica surfaces, particularlywhere the silica surface is incorporated into a fiber optic probesubsequently used for remote ion detection or quantitation.

In a particular aspect, the reactive group is a photoactivatable groupsuch that the group is only converted to a reactive species afterillumination with an appropriate wavelength. An appropriate wavelengthis generally a UV wavelength that is less than 400 nm. This methodprovides for specific attachment to only the target molecules, either insolution or immobilized on a solid or semi-solid matrix.Photoactivatable reactive groups include, without limitation,benzophenones, aryl azides and diazirines.

Preferably, the reactive group is a photoactivatable group, succinimidylester of a carboxylic acid, a haloacetamide, haloalkyl, a hydrazine, anisothiocyanate, a maleimide group, an aliphatic amine, a silyl halide, acadaverine or a psoralen. More preferably, the reactive group is asuccinimidyl ester of a carboxylic acid, a maleimide, an iodoacetamide,or a silyl halide. In a particular embodiment the reactive group is asuccinimidyl ester of a carboxylic acid, a sulfonyl halide, atetrafluorophenyl ester, an iosothiocyanates or a maleimide.

In another exemplary embodiment, the present compound is covalentlybound to a carrier molecule. If the compound has a reactive group, thenthe carrier molecule can alternatively be linked to the compound throughthe reactive group. The reactive group may contain both a reactivefunctional moiety and a linker, or only the reactive functional moiety.

A variety of carrier molecules are useful in the present invention.Exemplary carrier molecules include antigens, steroids, vitamins, drugs,haptens, metabolites, toxins, environmental pollutants, amino acids,peptides, proteins, nucleic acids, nucleic acid polymers, carbohydrates,lipids, and polymers. In exemplary embodiment, at least one memberselected from R^(b), R^(c), R^(d1), R^(d2), R^(e), R^(f), R^(h), R^(i),R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, or R¹⁰ comprises a carrier molecule.Preferably, at least one of R^(b), R^(c), R^(e), R^(f), R^(h), R^(i),R², R³, R⁴, R⁵, R⁶, or R⁷ comprises a carrier molecule or is attached toa carrier molecule. Alternatively, if the present compound comprises areactive group or solid support a reactive group may be covalentlyattached independently to those substituents, allowing for furtherconjugation to a reactive group, carrier molecule or solid support.

In an exemplary embodiment, the carrier molecule comprises an aminoacid, a peptide, a protein, a polysaccharide, a nucleoside, anucleotide, an oligonucleotide, a nucleic acid, a hapten, a psoralen, adrug, a hormone, a lipid, a lipid assembly, a synthetic polymer, apolymeric microparticle, a biological cell, a virus and combinationsthereof. In another exemplary embodiment, the carrier molecule isselected from a hapten, a nucleotide, an oligonucleotide, a nucleic acidpolymer, a protein, a peptide or a polysaccharide. In a preferredembodiment the carrier molecule is amino acid, a peptide, a protein, apolysaccharide, a nucleoside, a nucleotide, an oligonucleotide, anucleic acid, a hapten, a psoralen, a drug, a hormone, a lipid, a lipidassembly, a tyramine, a synthetic polymer, a polymeric microparticle, abiological cell, cellular components, an ion chelating moiety, anenzymatic substrate or a virus. In another preferred embodiment, thecarrier molecule is an antibody or fragment thereof, an antigen, anavidin or streptavidin, a biotin, a dextran, an antibody bindingprotein, a fluorescent protein, agarose, and a non-biologicalmicroparticle. Typically, the carrier molecule is an antibody, anantibody fragment, antibody-binding proteins, avidin, streptavidin, atoxin, a lectin, or a growth factor. Preferred haptens include biotin,digoxigenin and fluorophores.

Antibody binging proteins include, but are not limited to, protein A,protein G, soluble Fc receptor, protein L, lectins, anti-IgG, anti-IgA,anti-IgM, anti-IgD, anti-IgE or a fragment thereof.

In an exemplary embodiment, the enzymatic substrate is selected from anamino acid, peptide, sugar, alcohol, alkanoic acid, 4-guanidinobenzoicacid, nucleic acid, lipid, sulfate, phosphate, —CH₂OCOalkyl andcombinations thereof. Thus, the enzyme substrates can be cleave byenzymes selected from the group consisting of peptidase, phosphatase,glycosidase, dealkylase, esterase, guanidinobenzotase, sulfatase,lipase, peroxidase, histone deacetylase, endoglycoceramidase,exonuclease, reductase and endonuclease.

In another exemplary embodiment, the carrier molecule is an amino acid(including those that are protected or are substituted by phosphates,carbohydrates, or C₁ to C₂₂ carboxylic acids), or a polymer of aminoacids such as a peptide or protein. In a related embodiment, the carriermolecule contains at least five amino acids, more preferably 5 to 36amino acids. Exemplary peptides include, but are not limited to,neuropeptides, cytokines, toxins, protease substrates, and proteinkinase substrates. Other exemplary peptides may function as organellelocalization peptides, that is, peptides that serve to target theconjugated compound for localization within a particular cellularsubstructure by cellular transport mechanisms. Preferred protein carriermolecules include enzymes, antibodies, lectins, glycoproteins, histones,albumins, lipoproteins, avidin, streptavidin, protein A, protein G,phycobiliproteins and other fluorescent proteins, hormones, toxins andgrowth factors. Typically, the protein carrier molecule is an antibody,an antibody fragment, avidin, streptavidin, a toxin, a lectin, or agrowth factor. Exemplary haptens include biotin, digoxigenin andfluorophores.

In another exemplary embodiment, the carrier molecule comprises anucleic acid base, nucleoside, nucleotide or a nucleic acid polymer,optionally containing an additional linker or spacer for attachment of afluorophore or other ligand, such as an alkynyl linkage (U.S. Pat. No.5,047,519), an aminoallyl linkage (U.S. Pat. No. 4,711,955) or otherlinkage. In another exemplary embodiment, the nucleotide carriermolecule is a nucleoside or a deoxynucleoside or a dideoxynucleoside.

Exemplary nucleic acid polymer carrier molecules are single- ormulti-stranded, natural or synthetic DNA or RNA oligonucleotides, orDNA/RNA hybrids, or incorporating an unusual linker such as morpholinederivatized phosphates (AntiVirals, Inc., Corvallis Oreg.), or peptidenucleic acids such as N-(2-aminoethyl)glycine units, where the nucleicacid contains fewer than 50 nucleotides, more typically fewer than 25nucleotides.

In another exemplary embodiment, the carrier molecule comprises acarbohydrate or polyol that is typically a polysaccharide, such asdextran, FICOLL, heparin, glycogen, amylopectin, mannan, inulin, starch,agarose and cellulose, or is a polymer such as a poly(ethylene glycol).In a related embodiment, the polysaccharide carrier molecule includesdextran, agarose or FICOLL.

In another exemplary embodiment, the carrier molecule comprises a lipid(typically having 6-25 carbons), including glycolipids, phospholipids,and sphingolipids. Alternatively, the carrier molecule comprises a lipidvesicle, such as a liposome, or is a lipoprotein (see below). Somelipophilic substituents are useful for facilitating transport of theconjugated dye into cells or cellular organelles.

Alternatively, the carrier molecule is cells, cellular systems, cellularfragments, or subcellular particles. Examples of this type of conjugatedmaterial include virus particles, bacterial particles, virus components,biological cells (such as animal cells, plant cells, bacteria, oryeast), or cellular components. Examples of cellular components that canbe labeled, or whose constituent molecules can be labeled, include butare not limited to lysosomes, endosomes, cytoplasm, nuclei, histones,mitochondria, Golgi apparatus, endoplasmic reticulum and vacuoles.

In another embodiment the carrier molecule is a metal chelating moiety.While any chelator that binds a metal ion of interest and gives a changein its fluorescence properties is a suitable conjugate, preferred metalchelating moieties are crown ethers, including diaryldiaza crown ethers,as described in U.S. Pat. No. 5,405,975 to Kuhn et al. (1995);derivatives of 1,2-bis-(2-aminophenoxyethane)-N,N,N′,N′-tetraacetic acid(BAPTA), as described in U.S. Pat. No. 5,453,517 to Kuhn et al. (1995)(incorporated by reference) and U.S. Pat. No. 5,049,673 to Tsien et al.(1991); derivatives of 2-carboxymethoxy-aniline-N,N-diacetic acid(APTRA), as described by Ragu et al., Am. J. Physiol., 256: C540 (1989);and pyridyl-based and phenanthroline metal ion chelators, as describedin U.S. Pat. No. 5,648,270 to Kuhn et al. (1997).

Fluorescent conjugates of metal chelating moieties possess utility asindicators for the presence of a desired metal ion. While fluorescention-indicators are known in the art, the incorporation of thefluorinated fluorogenic and fluorescent compounds of the presentinvention imparts the highly advantageous properties of the instantfluorophores onto the resulting ion indicator.

The ion-sensing conjugates of the invention are optionally prepared inchemically reactive forms and further conjugated to polymers such asdextrans to improve their utility as sensors as described in U.S. Pat.Nos. 5,405,975 and 5,453,517.

In another exemplary embodiment, the carrier molecule non-covalentlyassociates with organic or inorganic materials. Exemplary embodiments ofthe carrier molecule that possess a lipophilic substituent can be usedto target lipid assemblies such as biological membranes or liposomes bynon-covalent incorporation of the dye compound within the membrane,e.g., for use as probes for membrane structure or for incorporation inliposomes, lipoproteins, films, plastics, lipophilic microspheres orsimilar materials.

In an exemplary embodiment, the carrier molecule comprises a specificbinding pair member wherein the present compounds are conjugated to aspecific binding pair member and are used to detect an analyte in asample. Alternatively, the presence of the labeled specific binding pairmember indicates the location of the complementary member of thatspecific binding pair; each specific binding pair member having an areaon the surface or in a cavity which specifically binds to, and iscomplementary with, a particular spatial and polar organization of theother. Exemplary binding pairs are set forth in Table 2.

TABLE 2 Representative Specific Binding Pairs antigen antibody biotinavidin (or streptavidin or anti-biotin) IgG* protein A or protein G drugdrug receptor folate folate binding protein toxin toxin receptorcarbohydrate lectin or carbohydrate receptor peptide peptide receptorprotein protein receptor enzyme substrate enzyme DNA (RNA) cDNA (cRNA)†hormone hormone receptor ion chelator antibody antibody-binding proteins*IgG is an immunoglobulin †cDNA and cRNA are the complementary strandsused for hybridization

In an exemplary embodiment, the present compounds of the invention arecovalently bonded to a solid support. The solid support may be attachedto the compound or through a reactive group, if present, or through acarrier molecule, if present. Even if a reactive group and/or a carriermolecule are present, the solid support may be attached through the A, Lor B moiety. In exemplary embodiment, at least one member selected fromR^(b), R^(c), R^(d1), R^(d2), R^(e), R^(h), R^(i), R², R³, R⁴, R⁵, R⁶,R⁷, R⁸, R⁹, or R¹⁰ comprises a solid support. Preferably, at least oneof R^(b), R^(c), R^(e), R^(f), R^(h), R^(i), R², R³, R⁴, R⁵, R⁶, or R⁷comprises a solid support or is attached to a solid support.Alternatively, if the present compound comprises a carrier molecule orreactive group a solid support may be covalently attached independentlyto those substituents, allowing for further conjugation to a anotherdye, carrier molecule or solid support.

A solid support suitable for use in the present invention is typicallysubstantially insoluble in liquid phases. Solid supports of the currentinvention are not limited to a specific type of support. Rather, a largenumber of supports are available and are known to one of ordinary skillin the art. Thus, useful solid supports include solid and semi-solidmatrixes, such as aerogels and hydrogels, resins, beads, biochips(including thin film coated biochips), microfluidic chip, a siliconchip, multi-well plates (also referred to as microtitre plates ormicroplates), membranes, conducting and nonconducting metals, glass(including microscope slides) and magnetic supports. More specificexamples of useful solid supports include silica gels, polymericmembranes, particles, derivatized plastic films, glass beads, cotton,plastic beads, alumina gels, polysaccharides such as Sepharose,poly(acrylate), polystyrene, poly(acrylamide), polyol, agarose, agar,cellulose, dextran, starch, FICOLL, heparin, glycogen, amylopectin,mannan, inulin, nitrocellulose, diazocellulose, polyvinylchloride,polypropylene, polyethylene (including poly(ethylene glycol)), nylon,latex bead, magnetic bead, paramagnetic bead, superparamagnetic bead,starch and the like.

In some embodiments, the solid support may include a solid supportreactive functional group, including, but not limited to, hydroxyl,carboxyl, amino, thiol, aldehyde, halogen, nitro, cyano, amido, urea,carbonate, carbamate, isocyanate, sulfone, sulfonate, sulfonamide,sulfoxide, etc., for attaching the compounds of the invention. Usefulreactive groups are disclosed above and are equally applicable to thesolid support reactive functional groups herein.

A suitable solid phase support can be selected on the basis of desiredend use and suitability for various synthetic protocols. For example,where amide bond formation is desirable to attach the compounds of theinvention to the solid support, resins generally useful in peptidesynthesis may be employed, such as polystyrene (e.g., PAM-resin obtainedfrom Bachem Inc., Peninsula Laboratories, etc.), POLYHIPE™ resin(obtained from Aminotech, Canada), polyamide resin (obtained fromPeninsula Laboratories), polystyrene resin grafted with polyethyleneglycol (TentaGel™, Rapp Polymere, Tubingen, Germany),polydimethyl-acrylamide resin (available from Milligen/Biosearch,California), or PEGA beads (obtained from Polymer Laboratories).

Preparation of Conjugates

Conjugates of components (carrier molecules or solid supports), e.g.,drugs, peptides, toxins, nucleotides, phospholipids and other organicmolecules are prepared by organic synthesis methods using the reactivereporter molecules of the invention, are generally prepared by meanswell recognized in the art (Haugland, MOLECULAR PROBES HANDBOOK, supra,(2002)). Preferably, conjugation to form a covalent bond consists ofsimply mixing the reactive compounds of the present invention in asuitable solvent in which both the reactive compound and the substanceto be conjugated are soluble. The reaction preferably proceedsspontaneously without added reagents at room temperature or below. Forthose reactive compounds that are photoactivated, conjugation isfacilitated by illumination of the reaction mixture to activate thereactive compound. Chemical modification of water-insoluble substances,so that a desired compound-conjugate may be prepared, is preferablyperformed in an aprotic solvent such as dimethylformamide,dimethylsulfoxide, acetone, ethyl acetate, toluene, or chloroform.Similar modification of water-soluble materials is readily accomplishedthrough the use of the instant reactive compounds to make them morereadily soluble in organic solvents.

Preparation of peptide or protein conjugates typically comprises firstdissolving the protein to be conjugated in aqueous buffer at about.1-10mg/mL at room temperature or below. Bicarbonate buffers (pH about 8.3)are especially suitable for reaction with succinimidyl esters, phosphatebuffers (pH about 7.2-8) for reaction with thiol-reactive functionalgroups and carbonate or borate buffers (pH about 9) for reaction withisothiocyanates and dichlorotriazines. The appropriate reactive compoundis then dissolved in an aprotic solvent (usually DMSO or DMF) in anamount sufficient to give a suitable degree of labeling when added to asolution of the protein to be conjugated. The appropriate amount ofcompound for any protein or other component is convenientlypredetermined by experimentation in which variable amounts of thecompound are added to the protein, the conjugate is chromatographicallypurified to separate unconjugated compound and the compound-proteinconjugate is tested in its desired application.

Following addition of the reactive compound to the component solution,the mixture is incubated for a suitable period (typically about 1 hourat room temperature to several hours on ice), the excess compound isremoved by gel filtration, dialysis, HPLC, adsorption on an ion exchangeor hydrophobic polymer or other suitable means. The compound-conjugateis used in solution or lyophilized. In this way, suitable conjugates canbe prepared from antibodies, antibody fragments, avidins, lectins,enzymes, proteins A and G, cellular proteins, albumins, histones, growthfactors, hormones, and other proteins.

Conjugates of polymers, including biopolymers and other higher molecularweight polymers are typically prepared by means well recognized in theart (for example, Brinkley et al., Bioconjugate Chem., 3: 2 (1992)). Inthese embodiments, a single type of reactive site may be available, asis typical for polysaccharides) or multiple types of reactive sites(e.g. amines, thiols, alcohols, phenols) may be available, as is typicalfor proteins. Selectivity of labeling is best obtained by selection ofan appropriate reactive dye. For example, modification of thiols with athiol-selective reagent such as a haloacetamide or maleimide, ormodification of amines with an amine-reactive reagent such as anactivated ester, acyl azide, isothiocyanate or3,5-dichloro-2,4,6-triazine. Partial selectivity can also be obtained bycareful control of the reaction conditions.

When modifying polymers with the compounds, an excess of compound istypically used, relative to the expected degree of compoundsubstitution. Any residual, unreacted compound or a compound hydrolysisproduct is typically removed by dialysis, chromatography orprecipitation. Presence of residual, unconjugated dye can be detected bythin layer chromatography using a solvent that elutes the dye away fromits conjugate. In all cases it is usually preferred that the reagents bekept as concentrated as practical so as to obtain adequate rates ofconjugation.

In an exemplary embodiment, the conjugate of the invention is associatedwith an additional substance, that binds either to the reporter moleculeor the conjugated substance (carrier molecule or solid support) throughnoncovalent interaction. In another exemplary embodiment, the additionalsubstance is an antibody, an enzyme, a hapten, a lectin, a receptor, anoligonucleotide, a nucleic acid, a liposome, or a polymer. Theadditional substance is optionally used to probe for the location of thedye-conjugate, for example, as a means of enhancing the signal of thedye-conjugate.

Applications and Methods of Use

The present invention also provides methods of using the compoundsdescribed herein to detect peroxidase activity in a sample, directly orindirectly by the production of peroxide. The methods are illustrated bythe use of the compound of the invention to detect an active oxygenspecies, e.g. those of skill in the art will appreciate that this focusis for clarity of illustration and does not limit the scope of themethods in which the compounds of the invention find use.

In another embodiment, the present invention provides methods of usingthe compounds described herein to detect an analyte in a sample or as atracing or tracking reagent in a biological sample. Alternatively, thepresent compounds are also used to detect of monitor metabolic activityin a cell including cell viability and proliferation.

In one embodiment, for the detection of peroxidase activity, theselected methods of the invention exploit the facile oxidation/reductionchemistry of the compounds of the invention, relying on theinterconversion between a hydroxy derivative and the correspondingketone as shown below:

Thus, in a further aspect, there is provided a method for determiningthe presence or absence of peroxide in a sample. The method includes:

-   -   a) contacting the sample with a fluorogenic compound to prepare        a labeled sample, wherein the fluorogenic compound is according        to formula II;    -   b) incubating the labeled sample for a sufficient amount of time        to prepare an incubated sample, wherein the peroxide reacts with        the compound in the presence of a peroxidase to produce a        fluorescent product;    -   c) illuminating the incubated sample with an appropriate        wavelength to prepare an illuminated sample; and    -   d) observing the illuminated sample whereby the presence or        absence of the peroxide in the sample is determined.

The peroxidase may be an enzyme such as horseradish peroxidase or anenzyme that has peroxidase activity, but which is not generallyconsidered a peroxidase, such as cyclooxygenase. Typically, theperoxidase is horseradish peroxidase.

In certain embodiments, the peroxide detected is hydrogen peroxide, suchas that produced by horseradish peroxidase. In another embodiment, theperoxide is not hydrogen peroxide, such as the transient peroxideproduced by cyclooxygenase.

In another embodiment, the invention provides a modified version of themethod set forth above for detecting peroxide in a sample. In themodified method, the peroxide is generated by an enzymatic reaction,e.g., the oxidation of a substrate by an oxidase. The compound of theinvention is added to the assay mixture at any time prior to or duringthe generation of the peroxide. The reaction of the peroxide with theperoxidase can occur in the presence of the oxidase or other peroxidegenerating system. Thus, it is within the scope of the methods of theinvention to react the peroxide with the peroxidase essentiallysimultaneously with the generation of the peroxide by the oxidase.

Exemplary enzymes of use in the methods of the invention includehorseradish peroxidase as the required peroxidase; and oxidases such asglutamate oxidase, amine oxidase, choline oxidase, cholesterol oxidase,galactose oxidase, xanthine oxidase, uricase oxidase, pyruvate oxidase,glycerin-3-phosphate oxidase, acyl Co A oxidase, glycerol oxidase andglucose oxidase to generate peroxide.

As illustrated in FIG. 1, the compounds of the invention are of use todetect peroxide, e.g., hydrogen peroxide in a sample. The compounds ofthe invention provide enhanced fluorescent signal relative to theanalogous non-fluorinated compound. Moreover, the compound of theinvention provides a fluorescence signal that is more stable over aprolonged period in the presence of peroxide than is that of thecorresponding non-fluorinated compound.

The compounds of the invention are also of use to detect the presence ofan oxidase in a sample wherein the oxidase generates peroxide that isdetected in the presence of peroxides by a fluorogenic compound of thepresent invention. As shown in FIG. 2, an exemplary compound of theinvention and its non-fluorinated analogue are both oxidized tofluorescent species by the action of COX-2. The compound of theinvention provides a greater fluorescence signal intensity than isobserved for its non-fluorinated analogue.

In another example, a compound of the invention is utilized to detecthemoglobin in a sample. FIG. 3 shows that the fluorinated compound ofthe invention provides a fluorescent signal in the presence ofhemoglobin that is enhanced relative to that observed for thenon-fluorinated analogue.

In another example, a compound of the present invention is used todetect the activity of an acidic enzyme, phytase. This was an unexpectedresult and a clear advantage over known compounds for detecting peroxidein a sample, See, FIG. 10. This performance advantage of the presentcompounds is not confined to increased fluorescent signal (brightness)but also superior limit of detection as determined by Z-factoranalaysis, See

Example 42

In the case of the phytase assay Compound 4 is shown to have superiorresistance to bleaching due to over-oxidation, thus contributing to anextended dynamic range in comparison to Amplex Red reagent. Thisphenomenon was further tested where the activity of HRP was measured ina range of pH compared to Amplex Red Reagent, See FIGS. 6 and 9. Thepresent fluorinated compounds demonstrate the ability to detect peroxidein a broader pH range than Amplex Red Reagent, providing for the abilityof the present compounds to be used in acidic environments compared tothe known Amplex Red reagent.

In another example, a compound of the present invention is used for theindirect detection of lipase activity. In this instance, lipaseactivity, in cells, breaks down triglycerides into free fatty acids andglycerol. In an assay format glycerol kinase and glycerol phosphateoxidase is added wherein the glycerol phosphorylated the glycerol andthe glycerol oxidase oxidizes the phosphorylated glycerol producingH₂O₂. Thus with the addition of HRP the peroxidase is detected resultingin a correlation to the lipase activity of cells. This particular assayhas diagnostic applications wherein the effect of drugs and diet can beaccurately assessed for their affect on lipase activity, which plays arole in the degradation of unwanted triglycerides. FIG. 4 shows that thefluorinated compound of the invention provides a fluorescent signal thatis enhanced relative to that observed for the non-fluorinated analogue.Alternatively, an assay is designed as a more direct measure of lipaseactivity, wherein triglycerides are used instead of glycerol along withtriglyceride lipase, See Example 51.

In another embodiment, the peroxidase is covalently attached to acarrier molecule. In this instance, carrier molecules include, but arenot limited to, an amino acid, a peptide, a protein, a polysaccharide, anucleoside, a nucleotide, an oligonucleotide, a nucleic acid polymer, ahapten, a biotin-binding protein, a psoralen, a drug, a hormone, alipid, a lipid assembly, a synthetic polymer, a polymeric microparticle,a biological cell or a virus. In a further aspect, the carrier moleculesis an antibody or fragment thereof, an avidin or streptavidin, a biotin,a blood component protein, a dextran, an antibody-binding protein, afluorescent protein, a growth factor, a lectin, a lipopolysaccharide, amicroorganism, a metal binding protein, a metal chelating moiety, anon-biological microparticle, a peptide toxin, aphosphotidylserine-binding protein, a structural protein, asmall-molecule drug, or a tyramide.

Thus, the present invention provides a modified method for determiningthe presence or absence of an analyte in a sample, wherein the methodcomprises:

-   -   a) contacting the sample with a peroxidase enzyme covalently        attached to a carrier molecule to prepare a contacted sample,        wherein the carrier molecule directly or indirectly associates        with the analyte;    -   b) contacting the contacted sample with a fluorogenic compound        to prepare a labeled sample, wherein the fluorogenic compound is        according to formula II;    -   c) incubating the labeled sample for a sufficient amount of time        to prepare an incubated sample, wherein the peroxide reacts with        the compound in the presence of a peroxidase to produce a        fluorescent product;    -   d) illuminating the incubated sample with an appropriate        wavelength to prepare an illuminated sample; and    -   e) observing the illuminated sample whereby the presence or        absence of the analyte in the sample is determined.

FIG. 5 demonstrates the ability of HRP conjugated to an anti-IgG to beused for the specific detection of c-reactive protein when a compound ofthe present invention in used as the fluorogenic compound. This figurealso demonstrates the increased fluorescent signal using Compound 4compared to non-fluorinated Amplex Red reagent. This methodology can beused to detect any specific analyte in an ELISA format with either theperoxidase conjugated to secondary antibody (or other antibody-bindingprotein) or primary antibody.

In other embodiments, the compounds according to Formula III(fluorescent dyes) are utilized for detection of an analyte in a sampleor as a tracing or tracking reagent in a biological sample. In general,these compounds of the present invention are utilized to stain a sampleto give a detectable optical response under desired conditions by firstpreparing a dye solution comprising a dye compound described above, at aconcentration sufficient to yield a detectable optical response underthe desired conditions. Specifically the methods for staining a samplecomprises:

-   -   a) contacting the sample with a fluorescent compound to prepare        a labeled sample, wherein the fluorescent compound is according        to formula III;    -   b) incubating the labeled sample for a sufficient amount of time        to prepare an incubated sample;    -   c) illuminating the incubated sample with an appropriate        wavelength to prepare an illuminated sample; and    -   d) observing the illuminated sample whereby the sample is        stained.

Optionally, the sample is washed to remove residual, excess or unbounddye. The dye compound typically forms a covalent or non-covalentassociation or complex with an element of the sample, or is simplypresent within the bounds of the sample or portion of the sample. Inthis instance the dye may be chemically reactive, conjugated to acarrier molecule, or conjugated to a solid support. Alternatively, thedye is not chemically reactive and is not conjugated to a carriermolecule or solid support. The dyes according to Formula III areintended to be used in any assay wherein a fluorescent compound findsuse. Furthermore, the compounds according to Formula III can be used asspectrally matched controls for the compounds according to Formula IIand IV. The following description of methods is meant to be illustrativebut not as a limitation for the intended use.

In one embodiment, the staining is used to determine a specifiedcharacteristic of the sample by further comparing the optical responsewith a standard or expected response. For example, the dye solution isused to monitor specific components of the sample with respect to theirspatial and temporal distribution in the sample. Alternatively, the dyepreferentially binds to a specific analyte in a sample, enabling theresearcher to determine the presence or quantity of that specificanalyte. In another embodiment, the dye is used to analyze the samplefor the presence of a mechanism that responds specifically to the dyecompound, such as oxidation or reduction. The desired analysis to beperformed determines the composition of the dye solution and chemicalnature of the dye itself. In another example, the dye is bound by anantibody directed against the dye itself, typically resulting thefluorescence quenching of the dye.

For biological applications, the dye solution is typically an aqueous ormostly aqueous solution that comprises one or more of the described dyecompounds. In one aspect of the invention, the dye solution comprises afluorinated fluorophore or fluorogen as described above; alternatively,the dye solution comprises a dye compound that is a reactive dye analog,as previously described.

In yet another exemplary embodiment, the dye solution includes a dyeconjugate as described above.

Solutions of the compounds of the invention are prepared according tomethods generally known in the art. As with related known fluorophoresand fluorogens, the dyes and dye-conjugates are generally soluble inwater and aqueous solutions having a pH greater than or equal to about6. Stock solutions of pure dyes, however, are typically dissolved inorganic solvent before diluting into aqueous solution or buffer.Preferred organic solvents are aprotic polar solvents such as DMSO, DMF,N-methylpyrrolidone, acetone, acetonitrile, dioxane, tetrahydrofuran andother nonhydroxylic, completely water-miscible solvents. The labelingsolution is prepared by diluting an aliquot of the stock solution intoaqueous buffer to the desired labeling concentration. For thosecompounds that comprise amine- and/or thiol-reactive groups it isnecessary to avoid buffers or mediums that contain amine- orthiol-containing molecules.

In general, the amount of dye or conjugate in the dye solution is theminimum amount required to yield detectable staining in the samplewithin a reasonable time, with minimal background fluorescence orundesirable staining. The exact concentration of dye or dye-conjugate tobe used is dependent upon the experimental conditions and the desiredresults, and optimization of experimental conditions is typicallyrequired to determine the best concentration of stain to be used in agiven application. The concentration of dye present in the dye solutiontypically ranges from nanomolar to micromolar. The requiredconcentration for the dye solution is determined by systematic variationin dye or dye-conjugate concentration until satisfactory dye staining isaccomplished. The starting ranges are readily determined from methodsknown in the art for use of similar compounds under comparableconditions for the desired optical response.

As stated above, the amount of dye added in the labeling solution is theminimum amount required to yield detectable cellular staining in thesample, without significant background fluorescence or staining of othernonselective structures such as organelles or cellular structures. Theeffects of overloading, or too much dye in a cell, may not beimmediately apparent. For example, peripheral blood lymphocytes respondnormally to concanavalin A when treated with up to 1 μM dye, but notmore than with more than 5 μM dye. All cell types are different and theresearch will determine the concentration that is appropriate for eachassay and cell type.

The amount of reagent required for staining eukaryotic cells depends onthe number of cells present, the permeability of the cell membrane tothe reagent and, in the case of the diaminodihydroxanthenes, the timerequired for intracellular metabolism to generate a fluorescent product.In the case of staining of tissues, the amount of reagent required mayalso vary with the accessibility of the reagent to the cells in thetissue. The required concentration for the labeling solution isdetermined by systematic variation in labeling concentration until asatisfactory fluorescent labeling is accomplished. Typically, the amountof fluorescent xanthylium reagent required is about 0.01 μM to about 50μM, more typically about 0.5 μM to about 25 μM. Lower concentrations inthe nanomolar range, such as from about 20 nM to about 500 nM, aretypically employed when staining organelles such as mitochondria.

Low concentrations of dye will require longer incubation times forequivalent fluorescent brightness to be reached. For example, stainingmitochondria incubated in 20 nM labeling solution will require about 1to 2 hours to reach an arbitrary level of fluorescent staining that isreached in about 30 minutes using a 50 nM labeling solution. Similarly,the level of staining reached in 30 minutes using a 75 nM labelingsolution of a diaminodihydroxanthene dye will require incubation for 90minutes in a 50 nM labeling solution.

In another aspect, the thiol-chemically reactive compounds uniformlystain the cytoplasm of live cells, See for example Compound 30. In thisapplication the compounds are well retained in living cells throughseveral generations. They are inherited by daughter cells after cellfusion and are not transferred to adjacent cells in a population. Inthis instance, the cells are loaded with the present compounds by addingthe labeling solution to the culture medium and then washing the cellsbriefly with fresh medium before analysis. The labeling solution isprepared by adding a stock solution to serum-free medium at a finalcontraction from about 0.1 μM to about 50 μM. For cells that are rapidlyproliferating or dividing the assay will generally require a higherconcentration of dye, typically from about 5 μM to about 50 μM, while aviability assay will typically require less dye, such as from about 0.1μM to about 10 μM. Testing of at least a ten-fold range of concentrationis recommended to determine the appropriate concentration for eachparticular assay.

Without wishing to be bound by a theory, it is likely that thethiol-reactive compounds are probably reacting with thiols in aglutathione S-transferase-mediated reaction. In many cells, glutathionelevels are high and glutathione transferase is ubiquitous. Thethiol-reactive compound is transformed into a cell-impermeantfluorescent dye-thioether adduct that can be fixed with aldehydefixatives, permitting long-term storage.

Following preparation of the labeling solution, the solution is combinedwith the sample being analyzed. In one embodiment, the dyes of thepresent invention are cell permeant, and can be introduced into thesample cell or cells by incubation of the cell or cells in the labelingsolution. Any other method of introducing the dye into the sample cell,such as microinjection of a labeling solution, scrape loading techniques(short mechanical disruption of the plasma membrane where the plasmamembrane is peeled away from the cytoplasm, the dye is perfused throughthe sample and the plasma membrane reassembled), or patch clamp methods(where an opening is maintained in the plasma membrane for long periods)can be used. Any other treatment that will permeabilize the plasmamembrane, such as electroporation, shock treatments or highextracellular ATP can be used to accelerate introduction of the dye intothe cellular cytoplasm. Typically the dye will be introduced into thesample cell by incubation in the labeling solution, or bymicroinjection. Preferably the dye is introduced in to the cell or cellsby incubation in the labeling solution. Microinjection of dye solutionis used when analysis of a single cell is desired, within a colony ofother sample cells.

The sample can be observed immediately after cellular or organellestaining is evident. After staining, the cells or isolated organelles ina sample can optionally be fixed. A number of fixatives and fixationconditions are suitable for practicing this invention. Useful fixativesinclude, but are not limited to, formaldehyde, paraformaldehyde,formalin, glutaraldehyde, cold methanol and 3:1 methanol:acetic acid.Typically, cell fixation is accomplished by incubating in a 3.7%solution of paraformaldehyde for about 15-30 minutes.

Fixation is optionally followed or accompanied by permeabilization, suchas with acetone, ethanol, DMSO or various detergents. Permeabilizationis utilized to allow bulky additional detection reagents to enter thecellular space (vida infra) that would ordinarily be impermeant withrespect to the cellular membrane. A large variety of fixatives, fixationconditions, and permeabilization agents are known in the art, and othermethods of fixing or permeabilizing sample cells in conjunction with thestains of the present invention will be obvious to one of ordinaryskill. Cells and organelles stained by dyes of the present inventionretain fluorescent staining even after fixation and extensivepermeabilization.

Optionally, the cells or isolated organelles are washed to improve theresults of the staining procedure. Washing the sample cell or cellsafter incubation in the labeling solution, or optionally after fixationor permeabilization, greatly improves the visualization of the cell andorganelles. This is largely due to the decrease in non-specificbackground fluorescence after washing. Satisfactory organellevisualization is possible without washing at low labeling concentrations(for example <50 nM).

In one aspect of the invention, the dye solution comprises a fluorinateddye that non-covalently associates with organic or inorganic materials.Exemplary embodiments of the fluorinated dyes that possess a lipophilicsubstituent can be used to stain lipid assemblies such as biologicalmembranes or liposomes by non-covalent incorporation of the dye compoundwithin the membrane, e.g. for use as probes for membrane structure orfor incorporation in liposomes, lipoproteins, films, plastics,lipophilic microspheres or similar materials.

The fluorinated compounds of the invention are useful as coloringagents, tracers for detecting the flow of fluids such as in angiography,and tracing of fluid flow through gap junctions of neurons according toprocedures known in the art for other dyes. The fluorinated dyes of theinvention are also useful in assays as haptens, according to knownmethods, because fluorination does not interfere with the recognition ofthe fluorophore by an anti-dye antibody.

The fluorinated reactive dye compounds of the invention can be used tocell surfaces, cell membranes or intracellular compartments such asorganelles, or in the cell's cytoplasm, See for example Compound 28.Certain reactive groups allow the retention of the fluorophore in cellsor organelles by reacting with cellular materials. In particular,haloalkyl-(Compound 30) or halomethylbenzamide-substituted fluorinatedfluorophores are used to react selectively with intracellular componentssuch as glutathione, or to retain the dye compounds within cells orwithin selected organelles where the dye compound is localized therein,according to methods previously described (U.S. Pat. No. 5,362,628 toHaugland et al, (1994); U.S. Pat. No. 5,576,424 to Mao et al. (1996) (incells); and U.S. Pat. No. 5,459,268 to Haugland et al. (1995) and U.S.Pat. No. 5,686,261 to Zhang et al. (1997) (in mitochondria).Polyfluoroaryl-substituted dye compounds are similarly retained incells, in part by covalent attachment. The reactive dyes are used tolocalize staining in a part of the sample, e.g., where the localizationof the corresponding functional group is indicative of a characteristicof the sample; or to retain the dye in a specific portion of the samplefor extended periods of time, e.g., to follow the stained portion of thesample through a period of time or sequence of events. Alternatively,the fluorinated reactive dyes are used according to this method to makedye-conjugates, as described above, that are separately useful forstaining.

In an exemplary embodiment in which the dye solution comprises adye-conjugate, the dye conjugate is a labeled member of a specificbinding pair, and is used as a fluorescent probe for the complementarymember of that specific binding pair, each specific binding pair memberhaving an area on the surface or in a cavity which specifically binds toand is complementary with a particular spatial and polar organization ofthe other. For example, Compound 28 can be used to form a dye-conjugateith an amine-containing molecule under conditions described above. Thefluorescent conjugate of a specific binding pair member is useful fordetecting and optionally quantifying the presence of the complementaryspecific binding pair member in a sample, by methods that are well knownin the art. Optionally, the complementary binding pair member is presentin an animal cell, plant cell, bacteria, yeast or virus. Alternatively,the complementary member is immobilized on a solid or semi-solidsurface, such as a polymer, glass slide, hydrogel, polymeric membrane orpolymeric particle (such as a polymeric bead). The dye-conjugate mayalso comprise a fluorinated dye in a blocked form wherein the block islater removed by the action of an enzyme or light, or the conjugate maybe one in which OR⁸ is OH, in which case detection is made followingoxidation of the probe to a fluorescent dye.

Representative specific binding pairs are shown in Table 2. Typically aspecific binding pair member conjugated to the dye is a ligand or areceptor. As used in this document, the term ligand means any organiccompound for which a receptor naturally exists or can be prepared. Areceptor is any compound or composition capable of recognizing a spatialor polar organization of a molecule, e.g. epitopic or determinant site.Ligands for which naturally occurring receptors exist include naturaland synthetic proteins, including avidin and streptavidin, antibodies,enzymes, and hormones; nucleotides and natural or syntheticoligonucleotides, including primers for RNA and single- anddouble-stranded DNA; lipids; polysaccharides and carbohydrates; and avariety of drugs, including therapeutic drugs and drugs of abuse andpesticides. The reactive dyes are used according to methods extensivelyknown in the art, to prepare antibody conjugates for use in microscopyand immunofluorescent assays and nucleotide or oligonucleotideconjugates for nucleic acid hybridization assays and nucleic acidsequencing (e.g., U.S. Pat. No. 5,332,666 to Prober, et al. (1994); U.S.Pat. No. 5,171,534 to Smith, et al. (1992); U.S. Pat. No. 4,997,928 toHobbs (1991); and WO Appl. 94/05688 to Menchen, et al., and a widevariety of other applications). Nucleotide conjugates are readilyincorporated by DNA polymerase and can be used for in situ hybridizationor other techniques.

The compounds of the invention are also of use in the numerousfluorescence polarization assays that use conjugates of fluorescent dyesto low molecular weight drugs and ligands, which will be improved by theuse of the fluorinated dye compounds of the invention, e.g., U.S. Pat.No. 4,420,568 to Wang (1983) and U.S. Pat. No. 4,510,251 to Kirkemo etal. (1985).

In those embodiments in which a fluorinated dye is conjugated to aspecific binding pair member that is a chelator of calcium, sodium,magnesium, potassium, or other biologically important metal ion, thedye-conjugate functions as an indicator of the ion, which indicators areoptionally further conjugated to a biological or plastic polymeraccording to methods known in the art; e.g., using fluorinated analogsof the compounds described in U.S. Pat. No. 5,453,517 to Kuhn, et al.(1995); U.S. Pat. No. 5,405,975 to Kuhn, et al. (1995). Alternatively,the dye itself acts as a pH indicator at pH values within about 1.5 pHunits of the individual dye's pKa. Typically the detectable opticalresponse of the ion indicators is a change in fluorescence.

In another exemplary embodiment, the dye compounds are fluorinated dyesthat are substrates for oxidative enzymes and other reactive oxidizingagents, particularly for peroxidase enzymes.

The fluorinated enzyme substrates optionally contain additionalsubstituents that provide additional advantages. Fluorinatedfluorophores modified to contain a lipophilic tail according to thesynthesis described in U.S. Pat. No. 5,208,148 to Haugland et al. (1993)(incorporated by reference), are useful for permeabilizing substratesfor intracellular enzymes.

In another exemplary embodiment of the invention, the compounds are usedto determine the efficiency of a cellular efflux pump of cells in asample. Preferably the dye compounds are diacetates or diphosphates. Thedye compound is used in the minimum concentration that gives adetectable fluorescence emission. Once the diacetate compounds areinside the cell, the blocking acetates are cleaved and the compoundbecomes highly fluorescent. The efficiency of the cellular efflux pumpof cells in the sample is determined by comparing the fluorescenceemission of cells in the sample with the fluorescence of cells having aknown efflux efficiency. Where the efflux pump is impaired, inhibited,or absent, the fluorescent compound is well retained in the cell; wherethe efflux pump is present and functioning, the fluorescence of thecells decreases markedly. The photostability of the present fluorinatedcompounds is advantageous for monitoring the time course offluorescence.

Another application where the enhanced photostability of the presentfluorinated dye compounds is particularly advantageous is use of the dyecompounds for tracing. One or more fluorinated dyes conjugated to abiologically compatible polymer, including amino acid polymers(typically proteins, including fluorescent proteins), carbohydratepolymers (typically dextrans), and polymeric microspheres (typicallypolystyrene) are readily prepared for use as tracers according tomethods known in the art.

The dye compounds are advantageously used to stain biological samples,i.e. samples that comprise biological components. In one embodiment ofthe invention, the sample comprises heterogeneous mixtures ofcomponents, including intact cells, cell extracts, bacteria, viruses,organelles, and mixtures thereof. In another aspect of the invention,the sample comprises a single component or homogeneous group ofcomponents, e.g. biological polymers such as amino acid polymers,nucleic acid polymers or carbohydrate polymers, or lipid membranecomplexes, whether the polymers are synthetic or natural.

The sample is typically stained by passive means, i.e., by incubationwith the dye solution. Any other method of introducing the dye into thesample, such as microinjection of a dye solution into a cell ororganelle, can be used to accelerate introduction of the dye into thesample. The dyes of the present invention are generally non-toxic toliving cells and other biological components, within the concentrationsof use.

The sample can be observed immediately after staining. The sample isoptionally combined with other solutions in the course of staining,including wash solutions, permeabilization and/or fixation solutions,and other solutions containing additional detection reagents. Washingfollowing staining generally improves the detection of the opticalresponse due to the decrease in non-specific background fluorescenceafter washing. Satisfactory visualization is possible without washing byusing lower labeling concentrations. A number of fixatives and fixationconditions suitable for practicing this invention are known in the art,including formaldehyde, paraformaldehyde, formalin, glutaraldehyde, coldmethanol and 3:1 methanol:acetic acid. Fixation is typically used topreserve cellular morphology and to reduce biohazards when working withpathogenic samples. Selected embodiments of the dyes described above arewell retained in cells, and sample cells stained with these dyes retainconsiderable fluorescent staining after fixation. Fixation is optionallyfollowed or accompanied by permeabilization, such as with acetone,ethanol, DMSO or various detergents, to allow bulky dye compounds,including dye-conjugates described above, to cross cell membranes,according to methods generally known in the art. The staining of thepresent invention is optionally combined with the use of an additionaldetection reagent that produces a detectable response due to thepresence of a specific cell component, intracellular substance, orcellular condition, according to methods generally known in the art.Where the additional detection reagent has spectral properties thatdiffer from those of the subject dye compounds, multi-color applicationsare possible.

The compounds of the invention are also of use to derivative lowmolecular weight compounds for their analysis by capillary zoneelectrophoresis (CZE), HPLC or other separation techniques.

In another embodiment, the compounds according to Formula IV are used tomeasure the viability of a cell culture. In this instance, thefluorogenic compounds are taken up by live cells and oxidized by themetabolic activity of the cells wherein fluorogenic compounds areconverted to fluorescent product.

Therefore, a method of the present invention comprises a) contacting thesample with a compound according to Formula IV to prepare a labeledsample; b) incubating the labeled sample for a sufficient amount of timeto prepare an incubated sample, wherein the compound is capable ofentering cells and being reduced to produce a fluorescent product; c)illuminating the incubated sample with an appropriate wavelength; and d)observing the illuminated sample whereby the metabolic activity isdetected and the resulting signal is proportional to the number ofviable cells present in the sample.

These compounds represent by Formula IV can be used in any method formeasuring metabolic activity of a cell including, but not limited tocell proliferation assays and cell viability assays. In the case of cellproliferation, the internal environment of proliferating cells is morereduced than that of non-proliferating cells. Specifically, the ratiosof NADPH/NADP, FADH/FAD, FMNH/FMN and NADH/NAD, increase duringproliferation. Compounds according to Formula IV, which can be reducedby these metabolic intermediates, are useful for monitoring cellproliferation because their reduction is accompanied by a measurableshift in color, See Example 46.

Illumination

At any time after or during an assay or staining procedure, the sampleis illuminated with a wavelength of light that results in a detectableoptical response, and observed with a means for detecting the opticalresponse. While the dye compounds are detectable colorimetrically, usingambient light, typically the dye compounds are detected by thefluorescence properties of the parent fluorophore. Upon illumination,such as by an ultraviolet or visible wavelength emission lamp, an arclamp, a laser, or even sunlight or ordinary room light, the dyecompounds, including dye compounds bound to the complementary specificbinding pair member, display intense visible absorption as well asfluorescence emission. Selected equipment that is useful forilluminating the dye-conjugates of the invention includes, but is notlimited to, hand-held ultraviolet lamps, mercury arc lamps, xenon lamps,argon lasers, laser diodes, and YAG lasers. These illumination sourcesare optionally integrated into laser scanners, fluorescence microplatereaders, standard or mini fluorometers, or chromatographic detectors.This colorimetric absorbance or fluorescence emission is optionallydetected by visual inspection, or by use of any of the followingdevices: CCD cameras, video cameras, photographic film, laser scanningdevices, fluorometers, photodiodes, quantum counters, epifluorescencemicroscopes, scanning microscopes, flow cytometers, fluorescencemicroplate readers, or by means for amplifying the signal such asphotomultiplier tubes. Where the sample is examined using a flowcytometer, a fluorescence microscope or a fluorometer, the instrument isoptionally used to distinguish and discriminate between the fluorinateddye compound and a second fluorophore with detectably different opticalproperties, typically by distinguishing the fluorescence response of thefluorinated dye-conjugate from that of the second fluorophore. Where thesample is examined using a flow cytometer, examination of the sampleoptionally includes isolation of particles within the sample based onthe fluorescence response of the dye compound by using a sorting device.A detectable optical response means a change in, or occurrence of, aparameter in a test system that is capable of being perceived, either bydirect observation or instrumentally. Such detectable responses includethe change in, or appearance of, color, fluorescence, reflectance,chemiluminescence, light polarization, light scattering, or x-rayscattering. Typically the detectable response is a change influorescence, such as a change in the intensity, excitation or emissionwavelength distribution of fluorescence, fluorescence lifetime,fluorescence polarization, or a combination thereof. The detectableoptical response may occur throughout the sample or in a localizedportion of the sample. The presence or absence of the optical responseafter the elapsed time is indicative of one or more characteristic ofthe sample. Comparison of the degree of staining with a standard orexpected response can be used to determine whether and to what degreethe sample possesses a given characteristic.

Sample Preparation

The end user will determine the choice of the sample and the way inwhich the sample is prepared. The sample includes, without limitation,any biological derived material or aqueous solution that is thought tocontain a target analyte, peroxide or an enzymatic system that producesperoxide. The samples may also include a reactive oxygen species, e.g.,peroxide, or a molecule or system, e.g., an enzymatic system thatproduces peroxide. Furthermore, the sample can include a buffer solutionthat contains a peroxidase, peroxide and fluorogenic compounds of thepresent invention to determine the ability of the sample to oxidize thecompound of the invention.

The sample can be a biological fluid such as whole blood, plasma, serum,nasal secretions, sputum, saliva, urine, sweat, transdermal exudates,cerebrospinal fluid, or the like. Biological fluids also include tissueand cell culture medium wherein an analyte of interest has been secretedinto the medium. Alternatively, the sample may be whole organs, tissueor cells from the animal. Examples of sources of such samples includemuscle, eye, skin, gonads, lymph nodes, heart, brain, lung, liver,kidney, spleen, thymus, pancreas, solid tumors, macrophages, mammaryglands, mesothelium, and the like. Cells include without limitationprokaryotic cells and eukaryotic cells that include primary cultures andimmortalized cell lines. Eukaryotic cells include without limitationovary cells, epithelial cells, circulating immune cells, β cells,hepatocytes, and neurons.

In an exemplary embodiment, for carrying out the assay, the enzymeconcentration is conveniently in the range of about 1 nM to 500 nM, moreusually in the range of about 25 to 250 nM. One may use an individualfluorogenic substrate or a mixture of substrates in order to determinethe substrate profile of the enzyme. The concentration range of eachsubstrate will be about 10 to 5000 μM, more usually 50 to 2000 μM.

Coenzyme, if any, is preferably present in excess, so as not be ratelimiting. Generally, with the concentrations of enzyme indicated above,the concentration of coenzyme will be at least about 0.1 mM, usually atleast about 1 mM and not more than about 25 mM. The coenzyme solutionshould be prepared freshly for each series of determinations.

Various buffers may be used that do not interfere with the enzymeactivity. These buffers include PBS, Tris, MOPS, HEPES, phosphate, etc.The pH will vary depending upon the particular monooxygenase beingassayed, generally being in the range of about 7.0-7.5, where the pH isselected to provide for at least about maximum enzyme activity. Theconcentration of buffer will be sufficient to prevent a significantchange in pH during the course of the reaction, generally being in therange of about 0.1 to 100 mM, more usually 0.5 to 50 mM.

The reaction time will usually be at least about 5 min, more usually atleast about 30 min and preferably not more than about 120 min, dependingupon the temperature, concentrations of enzyme and substrate, etc. Byusing a specific time period for the reaction or measuring thefluorescence at 2 different times, the rate of reaction can bedetermined for comparison with other determinations. The temperaturewill generally be in the range of about 20 to 50° C., more usually inthe range of about 25 to 40° C.

In certain instances, it may be advantageous to add a small amount of anon-ionic detergent to the sample. Generally the detergent will bepresent in from about 0.01 to 0.1 vol. %. Illustrative non-ionicdetergents include the polyoxyalkylene diols, e.g. Pluronics, Tweens,Triton X-100, etc.

After sufficient time for a detectable amount of product to form, thereaction is optionally quenched. Various quenching agents may be used,both physical and chemical. Conveniently, a small amount of awater-soluble inhibitor may be added, such as acetonitrile, DMSO, SDS,methanol, DMF, etc. The amount of inhibitor will vary with the nature ofthe inhibitor and may be determined empirically.

Kits

In another aspect, the present invention provides kits that include afluorogenic or fluorescent compound of the invention. The kit willgenerally also include instructions for using the compound of theinvention in one or more methods.

In an exemplary embodiment, the kit includes a reactive compound of theinvention and instructions for conjugating the dye to any substancepossessing an appropriate functional group, and optionally forrecovering or purifying the materials labeled thereby. This combinationof reactive dye and instructions therefore comprise a kit for labelingan appropriate substance. Selected appropriate substances include, butare not limited to, polymers of biological molecules (e.g. proteins,oligonucleotides or carbohydrates), polymeric resins and plastics (e.g.polystyrene), metals, glasses, and other organic or inorganicsubstances. The dyes of the present invention are well-suited for thepreparation of such a kit.

In another exemplary kit of the invention, the instructions provided arefor performing an assay that detects oxidative or reductive agents orconditions in a sample. For example, in one embodiment, directions areprovided for detecting a reactive oxygen species, or an enzyme,organism, or other agent that generates a reactive oxygen species in asample. In one aspect the kit further comprises an enzyme, a catalyst, areaction buffer, an enzyme substrate, a peroxide, a stop solution, or apositive control. In one aspect the enzyme has oxidase or peroxidaseactivity and the positive control is a compound according to formulaIII.

In another exemplary kit of the invention, the instructions provided arefor performing an ELISA wherein a peroxidase is conjugated to a carriermolecule and a compound according to formula II is provided as thefluorogenic substrate. In an exemplary embodiment the peroxidase is HRP.In one aspect the carrier molecule is an amino acid, a peptide, aprotein, a polysaccharide, a nucleoside, a nucleotide, anoligonucleotide, a nucleic acid polymer, a hapten, a biotin-bindingprotein, a psoralen, a drug, a hormone, a lipid, a lipid assembly, asynthetic polymer, a polymeric microparticle, a biological cell or avirus. In a further aspect, the carrier molecule is an antibody orfragment thereof, an avidin or streptavidin, a biotin, a blood componentprotein, a dextran, an IgG binding protein, a fluorescent protein, agrowth factor, a lectin, a lipopolysaccharide, a microorganism, a metalbinding protein, a metal chelating moiety, a non-biologicalmicroparticle, a peptide toxin, a phosphotidylserine-binding protein, astructural protein, a small-molecule drug, or a tyramide. In anotheraspect the carrier molecule specifically associates with the analyte,such as a primary antibody the binds the target analyte. Alternatively,the carrier molecule binds to the primary antibody, such as anti-IgG,anti-IgE or anti-IgA.

A detailed description of the invention having been provided above, thefollowing examples are given for the purpose of illustrating theinvention and shall not be construed as being a limitation on the scopeof the invention or claims.

EXAMPLES Example 1 Preparation of Compound 1

To a solution of 4-fluororesorcinol (W.-C. Sun, et al., J. Org. Chem.,62 (1997) 6469) in ethanol (100 mL) at 0° C. was added a solution of KOH(3.0 g, 53.3 mmol) in H₂O. Isoamyl nitrite (5.5 mL, 40.9 mmol) was thenadded dropwise and the combined solution was allowed to warm to roomtemperature and stirred for an additional 1 hr.

The solution was concentrated in vacuo to a thick oil. The oil wasdissolved in 1M HCl (200 mL), stirred for 1 hr and filtered to removeundissolved impurities. The filtrate was extracted with ethyl acetate(200 mL). The organic layer was washed with saturated NaCl (200 mL) anddried over anhydrous sodium sulfate. The filtrate was evaporated to ayellow-brown powder. The solid was stirred in dichloromethane for 48hrs, filtered and dried to a constant weight to give6-nitroso-4-fluororescorcinol (3.11 g, 84% yield), Compound 1. ¹H NMR(DMSO-d₆): 13.78 (1H, s, OH); 11.46 (1H, s, OH); 7.19 (1H, d, J=3 Hz);5.76 (1H, d, J=2 Hz).

Example 2 Preparation of Compound 2(2,8-Difluoro-3,7-dihydroxyphenoxazine Triethylammonium salt)

A mixture of 1 (2.56 g, 16.3 mmol) and 4-fluororesorcinol (2.07 g, 16.3mmol) in concentrated sulfuric acid (12 mL) was heated at 80° C. for 1hr. The reaction was cooled to room temperature and added to saturatedNaCl (150 mL) at 0° C. The mixture was stirred for 40 min and thenfiltered. The residual solids were dissolved in methanol (200 mL) andthe solution was adjusted to pH 9 with triethylamine. The resultantsolution was adsorbed on silica gel and concentrated to dryness invacuo. The material was purified on a silica gel column using 80%chloroform/19% methanol/1% triethylamine as the eluent. Purifiedmaterial was dried to a constant weight to give phenoxazine 2 (3.1 g,70% yield). ¹H NMR (DMSO-d₆) 9.21 (1H, broad s); 7.40 (2H, d, J=3 Hz);6.47 (2H, d, J=2); 3.10 (6H, q); 1.20 (9H, t).

Example 3 Preparation of Compound 3(2,8-Difluoro-3,7,10-triacetylphenoxazine)

To a mixture of tin (II) chloride (1.45 g, 7.6 mmol) and 2 (505 mg, 1.4mmol) in triethylamine (1.6 mL) was added acetic anhydride (10 mL, 105mmol). The mixture was heated to reflux for 20 min, cooled to roomtemperature and filtered. The filtrate was diluted with ethyl acetate(20 mL) and saturated bicarbonate solution (20 mL) and stirred at roomtemperature overnight. The organic layer was washed with 1M HCl (2×100mL) and then saturated NaCl (2×100 mL). The organic layer was dried overanhydrous sodium sulfate and evaporated to a brown oily semi-solid.Purification by silica gel chromatography with methylene chloride aseluent afforded 72 mg (14% yield) of pure material (3) after drying. ¹HNMR (CDCl₃): 7.34 (2H, d, J=3 Hz); 6.95 (2H, d, J=2 Hz,); 2.39 (3H, s);2.37 (6H, s)

Example 4 Preparation of compound 4(2,8-Difluoro-10-acetyl-3,7-dihydroxyphenoxazine)

To a solution of 3 (70 mg, 195 mmol) in CH₂Cl₂ and CH₃OH (10 mL, 1:1)was added sodium methoxide (1.1 eq) in methanol. The solution wasstirred at room temperature for 10 minutes. The solution was thenadsorbed onto silica gel and purified by silica gel chromatography using5% methanol in methylene chloride as the eluent to give 14 mg ofCompound 4. ¹H NMR (DMSO-d₆) 10.27 (2H, broad s, OH); 7.43 (2H, d, J=3Hz), 6.74 (2H, d, J=2 Hz); 2.25 (3H, s); ¹⁹F NMR (DMSO-d₆) −141.5 (2F,s).

Example 5 Preparation of Compound 5

A mixture of 2,4-difluororesorcinol (0.10 g, 0.68 mmol) and4-nitrosoresorcinol (95 mg, 0.68 mmol) was prepared in concentratedsulfuric acid (2 mL) on ice. The resulting mixture was heated to 80° C.with magnetic stirring for 36 hours, then cooled and diluted with water(40 mL). The resulting mixture was extracted with 3% methanol/ethylacetate (3×10 mL). The extract was dried over sodium sulfate, filteredthrough Celite and concentrated to give 5 as 70 mg of a red powder:fluorescence emission max 593 nm (excited at 560 nm, pH 9).

Example 6 Preparation of Compound 6

A mixture of 2-fluororesorcinol (0.10 g, 0.78 mmol) and4-nitrosoresorcinol (109 mg, 0.78 mmol) was prepared in concentratedsulfuric acid (2 mL). The resulting mixture was heated to 80° C. withmagnetic stirring for 36 hours, then cooled and diluted with water (50mL). The resulting mixture was extracted with ethyl acetate (3×30 mL).The extract was washed with water (lx 20 mL) and brine (1×20 mL), driedover sodium sulfate, and concentrated to give 6 as 40 mg of a redpowder: fluorescence emission max 597 nm (excited at 560 nm, pH 9).

Example 7 Preparation of Compound 7

A mixture of 4-fluororesorcinol (0.10 g, 0.78 mmol) and4-nitrosoresorcinol (0.11 g, 0.78 mmol) was prepared in concentratedsulfuric acid (3 mL). The resulting mixture was heated to 70° C. withmagnetic stirring for 24 hours, then cooled and diluted with water (60mL). The resulting mixture was extracted with ethyl acetate (2×40 mL)and ether (2×40 mL). The combined extracts were washed wither water(1×20 mL) and brine (1×20 mL), dried over sodium sulfate, andconcentrated to give 7 as 30 mg of a red solid: fluorescence emissionmax 586 nm (excited at 560 nm, pH 9).

Example 8 Preparation of Compound 8

A mixture of 5-fluororesorcinol (0.10 g, 0.78 mmol) and4-nitrosoresorcinol (0.11 g, 0.78 mmol) was prepared in concentratedsulfuric acid (3 mL). The resulting mixture was heated to 110° C. withmagnetic stirring for 24 hours, then cooled and diluted with water (50mL). The resulting mixture was extracted with ethyl acetate (2×20 mL).The extract was washed wither water (1×10 mL) and brine (1×10 mL), driedover sodium sulfate, and concentrated to give 8 as 5 mg of a redresidue: fluorescence emission max 585 nm (excited at 560 nm, pH 9).

Example 9 Preparation of Compound 9

A mixture of 2,4,5-trifluororesorcinol (0.10 g, 0.61 mmol) and4-nitrosoresorcinol (85 mg, 0.61 mmol) was prepared in concentratedsulfuric acid (2 mL). The resulting mixture was heated to 150° C. withmagnetic stirring for 4.5 hours, then cooled and diluted with 1:1brine/water (100 mL). The resulting mixture was extracted with ethylacetate (2×20 mL) and ether (1×20 mL). The combined extracts were washedwither water (1×10 mL) and brine (1×10 mL), filtered through Celite,dried over sodium sulfate, and concentrated to give Compound 9 as 79 mgof a red solid: emission max 589 nm (excited at 560 nm, pH 9).

Example 10 Photostability Study of Compounds 5-9

Compounds 5-9, fluorinated resorufin compounds, exhibited unexpectedlyimproved photostability over unsubstituted resorufin. For example,solutions of each were matched by optical density (0.03 at 560 nm inaqueous buffer at pH 9), then continuously irradiated in aspectrofluorimeter at 560 nm for 30 minutes. Over the duration of theexperiment, resorufin photobleached to a greater extent than any of thefluorinated analogs. The rank order of photostability of fluourinatedanalogs is indicated in Table 3.

TABLE 3 Relative Photostability Fluorescence remaining after irradiationCompound for 30 minutes at 560 nm Compound 9 98% Compound 6 97% Compound5 96% Compound 8 95% Compound 7 94% resorufin 91%

Example 11 Preparation of Compound 10

To a solution of 2-fluororesorcinol (1.5 g, 11.7 mmol) in ethanol (50mL) at 0° C. was added a solution of KOH (1.5 g, 26.6 mmol) in H₂O (5mL). Isoamyl nitrite (2.8 mL, 20.5 mmol) was then added dropwise and thecombined solution was allowed to warm to room temperature and stirredfor an additional 1 hr. The solution was concentrated in vacuo to athick oil. The oil was dissolved in 1M HCl (100 mL), stirred for 1 hrand filtered to remove un-dissolved impurities. The filtrate wasextracted with ethyl acetate (200 mL). The organic layer was washed withsaturated NaCl (200 mL) and dried over anhydrous sodium sulfate. Thefiltrate was evaporated to a yellow-brown powder. The solid was stirredin dichloromethane for 24 hrs, filtered and dried to a constant weightto give 10 (1.3 g).

Example 12 Preparation of Compound 11

A mixture of 6-nitroso-2-fluororesorcinol 10 (1.25 g, 8.1 mmol) and2-fluororesorcinol (1 g, 8.1 mmol) in concentrated sulfuric acid (6 mL)was heated at 80° C. for 1 hr. The reaction was cooled to roomtemperature and added to saturated NaCl (150 mL) at 0° C. The mixturewas stirred for 40 min and then filtered. The residual solids weredissolved in methanol (100 mL) and the solution was adjusted to pH 9with triethylamine. The resultant solution was adsorbed on silica geland concentrated to dryness in vacuo. The material was purified on asilica gel column using 80% chloroform/19% methanol/1% triethylamine asthe eluent. Purified material was dried to a constant weight to give thephenoxazine 11 (1.1 g).

Example 13 Preparation of Compound 12

2,4-Difluororesorcinol (2 g, 13.7 mmol) was converted to6-nitroso-2,4-difluororesorcinol 12 (1 g) using the procedure forpreparing 10.

Example 14 Preparation of Compound 13

A mixture of 6-nitroso-2,4-difluororesorcinol 12 (1 g, 5.34 mmol) and2,4-difluororesorcinol (0.78 g, 5.34 mmol) in concentrated sulfuric acid(6 mL) was heated at 80° C. for 1 hr. The reaction was cooled to roomtemperature and added to saturated NaCl (150 mL) at 0° C. The mixturewas stirred for 40 min and then filtered. The residual solids weredissolved in methanol (100 mL) and the solution was adjusted to pH 9with triethylamine. The resultant solution was adsorbed on silica geland concentrated to dryness in vacuo. The material was purified on asilica gel column using 80% chloroform/19% methanol/1% triethylamine asthe eluent. Purified material was dried to a constant weight to give thephenoxazine 13 (0.8 g).

Example 15 Preparation of Compound 14

**To a solution of 5-fluororesorcinol (1.5 g, 11.7 mmol) in ethanol (50mL) at 0° C. was added a solution of KOH (1.5 g, 26.6 mmol) in H₂O (5mL). Isoamyl nitrite (2.8 mL, 20.5 mmol) was then added dropwise and thecombined solution was allowed to warm to room temperature and stirredfor an additional 1 hr. The solution was concentrated in vacuo to athick oil. The oil was dissolved in 1M HCl (100 mL), stirred for 1 hrand filtered to remove insoluble impurities. The filtrate was extractedwith ethyl acetate (200 mL). The organic layer was washed with saturatedNaCl (200 mL) and dried over anhydrous sodium sulfate. The filtrate wasevaporated to a yellow-brown powder. The solid was stirred indichloromethane for 24 hrs, filtered and dried to a constant weight togive 14 (1.6 g).

Example 16 Preparation of Compound 15

A mixture of 6-nitroso-S-fluororesorcinol 14 (1.25 g, 8.1 mmol) and5-fluororesorcinol (1 g, 8.1 mmol) in concentrated sulfuric acid (6 mL)was heated at 80° C. for 1 hr. The reaction was cooled to roomtemperature and added to saturated NaCl (150 mL) at 0° C. The mixturewas stirred for 40 min and then filtered. The residual solids weredissolved in methanol (100 mL) and the solution was adjusted to pH 9with triethylamine. The resultant solution was adsorbed on silica geland concentrated to dryness in vacuo. The material was purified on asilica gel column using 80% chloroform/19% methanol/1% triethylamine asthe eluent. Purified material was dried to a constant weight to give thephenoxazine 15 (0.8 g).

Example 17 Preparation of Compound 16

To a solution of 4-chlororesorcinol (10 g, 69.4 mmol) in ethanol (50 mL)at 0° C. was added a solution of KOH (5.1 g, 76.3 mmol) in H₂O (10 mL).Isoamyl nitrite (10.3 mL, 90.2 mmol) was then added dropwise and thecombined solution was allowed to warm to room temperature and stirredfor an additional 1 hr. The solution was acidified to pH 2 with 10% HCl.The solid precipitate was collected by suction filtration and dried to aconstant weight to give 16 (5.5 g).

Example 18 Preparation of Compound 17

A mixture of 6-nitroso-4-chlororesorcinol 16 (0.5 g, 2.9 mmol) and4-chlororesorcinol (0.46 g, 3.19 mmol) in concentrated H₂SO₄ (3 mL) wasstirred at room temperature for 1 hr and then heated at 110° C. for 30minutes. After cooling to room temperature, the reaction mixture waspoured into ice-water. The precipitate was collected by suctionfiltration and dried to a constant weight to give 17 (0.6 g).

Example 19 Preparation of Compound 18

A suspension of 2,8-dichloro-3,7-dihydroxyphenoxazine 17 (0.2 g, 0.7mmol) and tin (II) chloride (0.269 g, 1.4 mmol) in acetic anhydride (10mL) was heated at 130° C. for 2 hrs and then cooled down to roomtemperature. The mixture was diluted with water and extracted with ethylacetate (2×50 mL). The combined organic layers were washed with 1M HCl(50 mL), saturated NaCl (50 mL) and dried with anhydrous sodium sulfate.The crude product was purified by silica gel column using chloroform aseluent to give 18 (80 mg).

Example 20 Preparation of Compound 19

To a solution of 2,8-chloro-3,7,10-triacetylphenoxazine 18 (80 mg, 0.2mmol) in CH₂Cl₂ and CH₃OH (4 mL, 1:1) was added sodium methoxide (1.1eq) in methanol. The solution was stirred at room temperature for 10minutes. The solution was then adsorbed on silica gel and purified bysilica gel chromatography using 5% methanol in methylene chloride as theeluent to give 19 (14 mg).

Example 21 Preparation of Compound 20

A suspension of 6-nitroso-4-chlororesorcinol 16 (0.5 g, 2.9 mmol) and2-carboxyresorcinol (0.445 g, 2.9 mmol) in concentrated sulfuric acid (3mL) was heated at 110° C. for 30 minutes. After cooling down to roomtemperature, the mixture was poured into ice water and the product wascollected by suction filtration and dried to constant weight to give 20(0.25 g).

Example 22 Preparation of Compound 21

To a suspension of 2-chloro-6-carboxy-3,7-dihydroxyphenoxazine 20 (0.5g, 1.95 mmol) and succinimidyl trifluoroacetate (0.49 g, 2.34 mmol) indry THF (10 mL) was added pyridine (0.32 mL, 2.34 mmol). The mixture wasstirred at room temperature overnight and concentrated to dryness. Theresidue was suspended in ethyl acetate and stirred at room temperaturefor 1 h. The precipitate was collected by suction filtration and driedto a constant weight to give 21 (0.35 g), used for the next step withoutfurther purification.

Example 23 Preparation of Compound 22

To a solution of 2-fluoro-8-dodecylresorufin (0.4 g, 1 mmol) in TFA (10mL) was added hydrogen peroxide-urea (1.5 g, 15 mmol). The mixture wasstirred at room temperature for 5 h and then was poured into water. Theaqueous layer was extracted with ethyl acetate (2×50 mL). The combinedorganic layers were washed with water (30 mL), saturated NaCl (30 mL)and dried with anhydrous Na₂SO₄. The crude product was purified bycolumn chromatography on silica gel using CHCl₃ and 2% MeOH in CHCl₃ aseluents to give 22 (30 mg).

Example 24 Preparation of Compound 23

To a 100 mL flask, methyl 4-bromobenzoate (1.42 g),2,4-dimethoxybenzeneboronic acid (1.3 g), Pd (OAc)₂ (100 mg), K₂CO₃ (1.9g) and tetrabutylammonium bromide (3.54 g) were added. The flask wasflushed with N₂ and sealed with a rubber septum. Water (50 mL) was addedwith a syringe, and the resulting suspension was stirred anddeoxygenated with N₂ at room temperature. The mixture was stirred andheated for 40 min. at 80° C. under N₂, cooled to room temperature, andacidified to pH 1 with concentrated HCl. The precipitate (Pd) wasfiltered off and washed with EtOAc. The filtrate was extracted withEtOAc, washed with saturated brine, dried over anhydrous Na₂SO₄ andconcentrated to give crude product 23.

Example 25 Preparation of Compound 24

To the crude 23 in a flask, 48% HBr in water (20 mL) and AcOH (20 mL)were added and the mixture was refluxed for 3 h under N₂. The resultingmixture was cooled to room temperature and poured into water (200 mL).The solution was extracted with EtOAc. The EtOAC layer was washed withbrine and dried over anhydrous Na₂SO₄. The EtOAc extract wasconcentrated in vacuo and purified on a silica gel column using 10:1CHCl₃/MeOH and 5:1 CHCl₃/MeOH as eluents to give compound 24 (0.97 g).

Example 26 Preparation of Compound 25

To compound 24 (0.97 g) in concentrated H₂SO₄ (20 mL) under N₂ was added4-fluoro-6-nitrosoresorcinol (0.59 g). The mixture was heated for 2 h at80° C. under N₂ and then poured into crushed ice/water. The suspensionwas extracted with EtOAc. The EtOAc layer was washed with brine anddried over anhydrous Na₂SO₄. The EtOAc extract was concentrated in vacuoand purified on a silica gel column using CHCl₃, 10:1 CHCl₃/MeOH and500:100:1 CHCl₃/MeOH/AcOH as eluents to give compound 25 (0.83 g).

Example 27 Preparation of Compound 26

To compound 25 (1.0 g) in AcOH (30 mL) and triethylamine (5 mL), tin(II) chloride dihydrate (5.0 g) was added and refluxed for 1 h. Theresulting reaction mixture was cooled to 70° C. and then diluted withEtOAc (200 mL). The mixture was cooled to room temperature and pouredinto crushed ice/water. The precipitate was filtered off and washed withEtOAc. The filtrate was extracted with EtOAc. The EtOAc layer was washedwith saturated brine, dried over anhydrous Na₂SO₄ and concentrated todryness. The crude product was purified on a silica gel column usingCHCl₃, 100:1 CHCl₃/MeOH and 50:1 CHCl₃/MeOH as eluents to give compound26 (214 mg).

Example 28 Preparation of Compound 27

To compound 26 (55 mg) in MeCN (10 mL)/MeOH (3 mL), K₂CO₃ (140 mg) wasadded and stirred for 2 h at room temperature under N₂ in the dark. Theresulting mixture was diluted with water (100 mL) and acidified withAcOH to pH 2. The solution was extracted with EtOAc. The EtOAc layer waswashed with brine and dried over anhydrous Na₂SO₄. The EtOAc extract wasconcentrated in vacuo and purified on a silica gel column using 10:1CHCl₃/MeOH and 5:1 CHCl₃/MeOH as eluents to give compound 27 (16 mg).

Example 29 Preparation of Compound 28

To compound 27 (50 mg) in dry DMF (5 mL) under N₂ in the dark, EDC (30mg) and N-hydroxysuccinimide (18 mg) were added and the mixture wasstirred for 5 h. The solvent was removed in vacuo and the residue waspurified on a short silica gel column under N₂ in the dark using 10:1CHCl₃/MeOH and 5:1 CHCl₃/MeOH as eluents to give compound 28 (32 mg)

Example 30 Preparation of Compound 29

To compound 26 (20 mg) in dry THF (6 mL) at 0° C., ethyl chloroformate(0.05 mL) and triethylamine (0.05 mL) were added. The mixture wasstirred for 1 h at RT. The white precipitate was filtered off, andwashed with dry THF. The filtrate was concentrated to dryness. Theresidue was re-dissolved in dry THF (6.0 mL), and then NaBH₄ (100 mg) inEtOH (1.0 mL) was added at 0° C. The mixture was stirred for 3 h at roomtemperature and then NaOH (1.0 N, 5.0 mL) was added. The solution washeated for 4 h at 60° C., and acidified to pH=3 with 10% HCl. Thesolution was diluted with water (100 mL) and extracted with EtOAc. TheEtOAc extract was washed with brine, dried over anhydrous Na₂SO₄ andconcentrated in vacuo. The residue was purified on a silica gel columnusing 20:1 CHCl₃/MeOH and 10:1 CHCl₃/MeOH as eluents to give compound 29(10 mg).

Example 31 Preparation of Compound 30

To compound 29 (10 mg) in THF (3 mL), con. HCl (10 mL) was added. Themixture was stirred for 2 days at room temperature. The solution wasconcentrated to dryness in vacuo. The residue was purified by apreparative silica gel TLC using 30:1 CHCl₃/MeOH as developing solventsto give compound 30 (6 mg).

Example 32 Preparation of Compound 31

To compound 27 (60 mg) in dry DMF (10 mL) under N₂ in the dark, EDC (30mg) and N-hydroxysuccinimide (18 mg) were added and the mixture wasstirred for 5 h at room temperature. To the resulting reaction mixture,N-(5-aminopentyl)biotinamide, trifluoroacetic acid salt (80 mg) andN(Pr-i)₂Et (0.05 mL) were added and the mixture was stirred at roomtemperature for 6 h. The solution was concentrated to about 3 mL andthen diluted with MeOH (10 mL). To the solution, K₂CO₃ (300 mg) in water(10 mL) was added and stirred at room temperature for 3 h under N₂ inthe dark. The mixture was poured into saturated brine (100 mL) andacidified with AcOH to pH 2. The acidified solution was extracted withEtOAc and the EtOAc layer washed with saturated brine and dried overanhydrous Na₂SO₄. The EtOAc extract was concentrated in vacuo andpurified on a silica gel column using 10:1 CHCl₃/MeOH and 5:1 CHCl₃/MeOHas eluents to give compound 31 (30 mg).

Example 33 Detection of H₂O₂ in the Presence of HRP in a Solution Assay

Amplex Red (Molecular Probes, Inc. A-12222) and Compound 4 wereresuspended to 10 mM in dry DMSO. H₂O₂ was serially diluted two-foldacross six rows of a 96-well microplate in 50 μl of 50 mM Tris, pH 7.5.50 μl of 100 μM Amplex Red+2 Units/ml HRP or 50 μl of 100 μM Compound4+2 Units/ml HRP were added to all wells and incubated at roomtemperature (˜25° C.) for twenty minutes. The resulting fluorescenceintensity was measured on a Victor² microplate reader (Wallac), 1read/well for 0.2 sec each at 10000V gain, excitation 535+/−17.5 nm,emission 590+/−17.5 nm.

Initially the fluorescence intensity of both dyes is relatively stablein the presence of high concentrations of H₂O₂, but after twenty minutesof incubation, there is a biphasic mode to the dilution series, wherebythe fluorescence has a peak at ˜40 μM H₂O₂, then quickly drops at 80-160μM H₂O₂ rising slowly at higher concentrations. See, FIG. 1

Example 34 Cyclooxygenase-2 assay

Amplex Red (Molecular Probes, Inc. A-12222) and Compound 4 wereresuspended to 10 mM in dry DMSO. Recombinant human cyclooxygenase-2(COX-2, from Cayman Chemical) was serially diluted (in triplicate foreach dye) two-fold in a 96-well microplate in 50 μl of 100 mM Tris, pH8.0. 10 μl of 20 μM hemin (a COX-2 cofactor) in 100 mM Tris, pH 8.0 wasadded to all wells. After a five minute incubation at room temperatureto allow the hemin to interact with the COX-2 enzyme, 40 μl of either125 μM Amplex Red+250 μM arachidonic acid or 125 μM Compound 4+250 μMarachidonic acid in 100 mM Tris, pH 8.0 was added to all wells. Thefinal reagent concentrations in the wells was zero to 50 Units/ml COX-2,2 μM hemin, 100 μM arachidonic acid, and 50 μM Amplex Red reagent orCompound 4 in 100 μl of 100 mM Tris, pH 8.0.

The resulting reactions were incubated at 37° C. in the dark for thirtyminutes. At that point, the resulting fluorescence was measured on aPerSeptive Biosystems CytoFluor 4000 microtiter plate reader(Framingham, Mass.). The excitation filter was 530 nm (+/−12.5 nm) andthe emission filter 590 nm (+/−17.5 nm), with a gain setting of 40.

As FIG. 2 shows, both dye reagents are oxidized to their fluorescentforms by COX-2. The dynamic range and sensitivity of both dyes issimilar with Compound 4 demonstrating a greater fluorescent intensitysignal. Error bars in the graph are one standard deviation from the meanof three measurements. See, FIG. 2

Example 35 Hemoglobin Assay

Amplex Red (Molecular Probes Inc. A-12222) and Compound 4 were eachresuspended to 10 mM in dry DMSO. Bovine hemoglobin (Sigma, catalog#H-2500) was serially diluted (in triplicate for each dye) two-fold in a96-well microplate in 50 μl of 50 mM Tris, pH 7.5. 50 μl of either 100μM Amplex Red+100 μM H₂O₂ or 100 μM Compound 4+100 μM H₂O₂ in 50 mMTris, pH 7.5 was added to all wells. The final reagent concentrations inthe wells was zero to 2000 ng/ml bovine hemoglobin, 50 μM H₂O₂, and 50μM Amplex Red or Compound 4 in 100 μl of 100 mM Tris, pH 7.5.

The resulting reactions were incubated at 37° C. in the dark for thirtyminutes. At that point, the resulting fluorescence was measured on aPerSeptive Biosystems CytoFluor 4000 microtiter plate reader(Framingham, Mass.). The excitation filter was 530 nm (+/−12.5 nm) andthe emission filter 590 nm (+/−17.5 nm), with a gain setting of 40.

As the FIG. 3 shows, both dye reagents are oxidized to their fluorescentforms by bovine hemoglobin. The dynamic range and sensitivity of bothdyes is similar, although Compound 4 is brighter. Error bars in thegraph are one standard deviation from the mean of three measurements.See, FIG. 3

Example 36 Glycerol Assay

Amplex Red (Molecular Probes Inc. A-12222) and Compound 4 were eachresuspended to 10 mM in dry DMSO. Glycerol (Sigma # G-7893) was seriallydiluted (in triplicate for each dye) two-fold in a 96-well microplate in50 μl of 50 mM Tris, pH 7.5. 50 μl of either 100 μM Amplex Red+2 Unit/mlHRP+2 U/ml Glycerokinase (Sigma # G-0774)+2 U/ml Glycerol 3-phosphateoxidase (Sigma # G-4388)+1 mM ATP or 100 μM Compound 4+2 Unit/ml HRP+2U/ml Glycerokinase (Sigma # G-0774)+2 U/ml Glycerol 1-phosphate oxidase(Sigma #G-4388)+1 mM ATP in 50 mM Tris, pH 7.5 was added to all wells.The final reagent concentrations in the wells was zero to 100 μMglycerol, 1 Unit/ml HRP+1 U/ml Glycerokinase+1 U/ml Glycerol 1-phosphateoxidase+0.5 mM ATP and either 50 μM Amplex Red or Compound 4 in 100 μlof 50 mM Tris, pH 7.5.

The resulting reactions were incubated at 37° C. in the dark for tenminutes. At that point, the resulting fluorescence was measured on aPerSeptive Biosystems CytoFluor 4000 microtiter plate reader(Framingham, Mass.). The excitation filter was 530 nm (+/−12.5 nm) andthe emission filter 590 nm (+/−17.5 nm), with a gain setting of 35.

As FIG. 4 shows, both dye reagents are oxidized to their fluorescentforms by the action of HRP and H₂O₂ (H₂O₂ generated by thecoupled-enzyme cascade). The dynamic range and sensitivity of both dyesis similar, although Compound 4 is brighter and hence a broader signalwindow. Error bars in the graph are one standard deviation from the meanof three measurements. See, FIG. 4

Example 37 Synthesis of Compound 32

To a 0.05 M solution of 2,7-difluororesorufin (Compound 2) intrifluoroacetic acid is added 15 equivalents of urea hydrogen peroxideaddition compound (percarbamide) at room temperature. The resultingsolution is stirred for 10 hours, or until TLC indicates reactioncompletion; on analytical TLC the resazurin Compound 32 has about ½ theR_(f) of the starting resorufin in chloroform/methanol mixtures. Thevolatiles are removed in vacuo, and the residue is diluted with waterand extracted with ethyl acetate (2×). The extract is dried over sodiumsulfate, filtered, and concentrated in vacuo to yield Compound 32 as ared solid.

This solid can be purified further by flash chromatography on mediumsilica gel using increasing amounts (up to 5%) of methanol inchloroform. Combine pure product fractions and concentrate in vacuo.

Example 38 C-Reactive Protein ELISA

Amplex Red (Molecular Probes Inc. A-12222) and Compound 4 were eachresuspended to 10 mM in dry DMSO. C-reactive protein (Fitzgerald#30-AC07) was serially diluted (in quadruplicate for each dye)three-fold in a 96-well ELISA microplate (Nunc #449824) in 100 μl of 50mM sodium phosphate, pH 7.4, 150 mM sodium chloride (PBS), and allowedto bind to solid-phase monoclonal mouse anti-human CRP (Fitzgerald#10-C33) for 1 hour at 25° C. All wells were washed three times in 200μl of PBS containing 0.1% Tween-20 (Aldrich #274348) and 100 μl of PBScontaining 50 ng/ml rabbit anti-human CRP (Calbiochem #235752) was addedto each well, and incubated at 25° C. for one hour. All wells were thenwashed three times in 200 μl of PBS containing 0.1% Tween-20, and 100 μlof 50 ng/mL goat anti-rabbit IgG-HRP conjugate (Molecular Probes #G-21234) was allowed to incubate at 25° C. in each well for 30 minutes.100 μl of either 50 μM Amplex Red+200 μM hydrogen peroxide (Aldrich#323381) or 50 μM Compound 4+200 μM hydrogen peroxide (Aldrich #323381)in 50 mM sodium phosphate, pH 7.4, 150 mM sodium chloride (PBS) wasadded to all wells. The final reagent concentration in the wells waszero to 6 ng C-reactive protein, 50 μM Amplex Red or Compound 4, 200 μMhydrogen peroxide in 100 μl of 50 mM sodium phosphate, pH 7.4, 150 mMsodium chloride.

The resulting reactions were incubated at 25° C. in a PerSeptiveBiosystems CytoFluor 4000 microtiter plate reader (Framingham, Mass.).The excitation filter was 530 nm (+/−12.5 nm) and the emission filter580 nm (+/−25 nm), with a gain setting of 35. Fluorescence was readevery five minutes for up to one hour.

As FIG. 5 shows, both dye reagents are oxidized to their fluorescentforms by the action of goat anti-rabbit IgG-HRP conjugate and H₂O₂. Thedynamic range and sensitivity of both dyes is similar, although Compound4 is brighter and hence a broader signal window. Error bars in the graphare one standard deviation from the mean of four measurements. See, FIG.5

Example 39 pH Tolerance Assay

Amplex Red (Molecular Probes Inc. A-12222) and Compound 4 were eachresuspended to 10 mM in dry DMSO. Hydrogen peroxide (Aldrich #323381)was serially diluted two-fold from 100 μM to zero in 100 μl of each ofeight 100 mM buffers of different pH:

TABLE 4 Buffer pH Sodium acetate (Sigma #S-8750) 5.0 MES (Sigma #M-3671) 6.0 MES (Sigma # M-3671) 6.5 MOPS (Sigma # M-1254) 7.0 MOPS(Sigma # M-1254) 7.5 Tris (Aldrich # 252859) 8.5 Borate (Sigma # S-9640)9.5 CAPS (Aldrich # 163767) 10.0

To each well, 100 μl of either 0.2 U/mL horseradish peroxidase (Sigma #P-8250)+100 μM Amplex Red (Molecular Probes Inc. A-12222) or 100 μMCompound 4 was added to begin the reaction.

The resulting reactions were incubated at 25° C. in a PerSeptiveBiosystems CytoFluor 4000 microtiter plate reader (Framingham, Mass.).The excitation filter was 530 nm (+/−12.5 nm) and the emission filter580 nm (+/−25 nm), with a gain setting of 35. Fluorescence was readevery five minutes for up to thirty minutes.

As the FIG. 6 shows, both dye reagents are oxidized to their fluorescentforms by peroxidase and H₂O₂. The sensitivity of both dyes is similar,but Compound 4 retains full fluorescence from pH 5 to 10 while AmplexRed has full fluorescence only from pH 6.5 to 7.5. See, FIG. 6

Example 40 Comparing Detection of LPS-Induced COX-2 Activity with AmplexRed Reagent and Compound 4

Two 100 mm plates of RAW 264.7 mouse macrophage cells (ATCC # TIB-71)were grown overnight to medium density in DMEM+10% FBS. The followingmorning the old media was removed and replaced with 12 ml fresh, warm,DMEM+10% FBS. To one plate, 12 μl of 100 μg/ml lipopolysaccaride (LPS)from E. coli strain 055:B5 (Sigma, catalog #L2880) in water was added.To the other plate, 12 μl of water alone was added. Both plates wereincubated for 8 hours, 37° C., 5% CO₂. The cells from each plate weregently resuspended into ice-cold 100 mM Tris, pH 7.5 to 4000 cells/0(counted by hemocytometer). Both batches of cells were lysed with aprobe sonicator for 40 seconds with a 30% duty cycle using power outputof 3 on a scale of 1 to 10. 50 μl of the resulting cell lysates wereadded to a 96-well microplate. 10 μl of 100 mM Tris, pH 7.5, +/−50 μMDuP-697 (Cayman Chemical catalog #70645), a COX-2 specific inhibitor,was added to all wells. The plate was incubated at room temperature(˜24° C.) for 10 minutes to allow the COX-2 inhibitor time to inhibitthe enzyme. 40 μl of 200 μM arachidonic acid (Cayman Chemical catalog#90010) and 100 μM dye (either Amplex Red or Compound 4) in 100 mM Tris,pH 7.5 was then added to the wells.

The final reagent concentrations in the wells was 200,000 lysed cellequivalents, +/−5 μM DuP-697, 80 μM arachidonic acid, and 40 μM AmplexRed or Compound 4 in 100 μl of 100 mM Tris, pH 7.5.

The resulting reactions were incubated at 37° C. in the dark for twentyminutes. At that point, the resulting fluorescence was measured on aPerSeptive Biosystems CytoFluor 4000 microtiter plate reader(Framingham, Mass.). The excitation filter was 530 nm (+/−12.5 nm) andthe emission filter 590 nm (+/−17.5 nm), with a gain setting of 50.

The graph of the resulting data demonstrates that both dyes can detectthe presence of an oxidizing enzyme that is stimulated by the 8 hour LPStreatment. Approximately half of this oxidation can be inhibited by theCOX-2-specific inhibitor DuP-697. Thus, both dyes can detect thepresence of induced COX-2 in mouse macrophages. However, compound 4demonstrates a stronger relative fluorescent signal, an improvement overthe known Amplex red reagent. See, FIG. 7.

Example 41 Detection of Acid Phosphatase (Phytase) Activity with AmplexRed and Compound 4 at pH 5.5

Phytases catalyze the sequential hydrolysis of phytate (myo-inositolhexakisphosphate; phytin; phytic acid) to less phosphorylatedmyo-inositol compounds and inorganic phosphate. This assay detectsphytase activity on the basis of measurement of phosphate release fromthe substrate phytic acid. In a series of linked enzymatic reactions,phytase catalyzes the release of inorganic phosphate from phytic acid;maltose phosphorylase (EC 2.4.1.8) converts maltose (in the presence ofP_(i)) to glucose 1-phosphate and glucose. Glucose oxidase (EC 1.1.3.4)converts the glucose to gluconolactone and H₂O₂. With horseradishperoxidase (HRP; EC 1.11.1.7) as a catalyst, the H₂O₂ reacts with thefluorogenic substrate (Amplex Red or Compound 4) to produce resorufin ordifluororesorufin.

A dilution series of a commercial preparation of phytase (Natuphos®10000L, BASF Wyandotte Corp, Wyandotte, Mich.; EC 3.1.3.8) was preparedin 0.1 M sodium acetate, pH 5.5. The enzyme dilutions were added to aCoStar 96-well round bottom plate in 50 μL aliquots.

Mixtures containing 4 units/mL maltose phosphorylase, 2 units/mL glucoseoxidase, 0.4 units/mL HRP, 2 mM phytic acid, 0.4 mM maltose, 0.1 Msodium acetate, pH 5.5, and 100 μM Amplex Red or 100 μM Compound 4 wereprepared, and reactions were initiated by addition of 50 μL of reagentmixture to the enzyme dilution series. Reactions were done intriplicate, for 60 minutes at 37° C. Fluorescence was measured with aCytoFluor® Series 4000 Multi-Well Plate Reader (PerSeptive Biosystems,Framingham, Mass.), 530+/−12.5 nm excitation, 580+/−25 nm emission. Thefluorescent signal was graphed as a function of phytase concentrationexpressed as international phytase units/mL (FTU/mL) (See, FIG. 8) andthe Z-factor (Table 5) was calculated according to Zhang et al. (1999).

TABLE 5 Z factor scores for data points shown in FIG. 8. Phytase, AmplexRed Compound 4 FTU/mL Z-factor Z-factor 0.1 −1.20 0.87 0.033 0.90 0.890.01 0.81 0.76 0.0033 0.13 0.76 0.001 0.30 0.64 0.00033 −1.74 0.09

As shown graphically, the signal strength and dynamic range under thesereaction conditions was significantly greater with Compound 4 as thereporting fluorogenic substrate. A Z-factor score of less than −1 orgreater than 1 indicates an assay values that do not differsignificantly or positively from the background value. With Compound 4the lower limit of detection of phytase activity in the assayed sampleis less than 0.00033 FTU/mL, and with Amplex Red the lower limit ofdetection is 0.001 FTU/mL.

Example 42 Detection of Horseradish Peroxidase Activity at Acidic pH:Comparison of Amplex Red and Compound 4

A dilution series of horseradish peroxidase (HRP; EC 1.11.1.7) wasprepared in 0.1 M sodium acetate, pH 5.5. The enzyme dilutions wereadded to a CoStar 96-well round bottom plate in 50 μL aliquots. Mixturescontaining 100 μM hydrogen peroxide (H₂O₂), 0.1 M sodium acetate, pH5.5, and 100 μM Amplex Red or 100 μM Compound 4 were prepared, andreactions were initiated by addition of 50 μL of reagent mixture to theenzyme dilution series. Reactions were done in triplicate, for 60minutes at 30° C. Fluorescence was measured with a CytoFluor® Series4000 Multi-Well Plate Reader (PerSeptive Biosystems, Framingham, Mass.),530+/−12.5 nm excitation, 580+/−25 nm emission. The fluorescent signalwas graphed as a function of HRP concentration (See, FIG. 9) and theZ-factor (Table 6) was calculated according to Zhang et al. (1999).

TABLE 6 Z factor scores for data points shown in FIG. 9. HRP, Amplex RedCompound 4 units/mL Z-factor Z-factor 0.25 0.85 0.89 0.125 0.69 0.810.063 0.51 0.90 0.032 2.14 0.89 0.016 1.30 0.72

As shown graphically, the signal strength and dynamic range under thesereaction conditions was significantly greater with Compound 4 as thereporting fluorogenic substrate. A Z-factor score of less than −1 orgreater than 1 indicates an assay values that do not differsignificantly or positively from the background value. With Compound 4the lower limit of detection of HRP activity in the assayed sample isless than 0.016 enzyme units/mL, and with Amplex Red the lower limit ofdetection is 0.032 units/mL.

Example 43 Detection of Myeloperoxidase Using Compound 4

Myeloperoxidase (Sigma # M-6908) was diluted to 1 unit/mL usingphosphate buffered saline (PBS, 50 mM sodium phosphate, pH 7.4, 150 mMsodium chloride). The solution was then serially diluted two fold usingthe same buffer. 50 μL of the preparations were added in a 96-wellmicroplate (Nunc #449824). 20 μL of 625 μM Compound 4 and 30 μl of 165μM hydrogen peroxide (Aldrich #323381) in PBS were added to all wells.The final reagent concentration in the wells was zero to 0.5 U/mLmyeloperoxidase, 125 μM Compound 4 and 50 μM hydrogen peroxide in 100 μlof PBS.

The resulting reactions were incubated at 37° C. in a PerSeptiveBiosystems CytoFluor 4000 microtiter plate reader (Framingham, Mass.).The excitation filter was 530 nm (+/−12.5 nm) and the emission filter580 nm (+/−25 nm), with a gain setting of 35. Fluorescence was readevery five minutes for up to 30 minutes.

As shown by the figure below, Compound 4 is oxidized to theirfluorescent forms by the action of myeloperoxidase, chloride and H₂O₂.The assay can detect myeloperoxidase down to 0.0078 U/mL which is about9.6 ng/mL. This limit of detection (LOD) is comparable with the resultsobtained from the most sensitive myeloperoxidase ELISA assay. Thedynamic range of the assay is between 0.0078 U/mL and 0.125 U/mL. To thebest of our knowledge this is the first time a fluorescent assay formyeloperoxidase has been presented. See, FIG. 10.

Example 44 Staining of Cells for Long Term Tracing of Living Cells UsingCompound 30

Grow cells in an appropriate culture medium. Adherent cells can be grownon coverslips inside Petri dishes filled with culture medium. Forexample, a calf pulmonary arterial endothelium (CPAE) cell line isobtained from American Type Culture Collection Co., Rockville Md. Thecells are maintained in a humidified atmosphere of 5% CO₂ in Dulbecco'smodified Eagle's medium (DMEM) supplemented with 10% calf serum, 50μg/mL gentamicin, 300 μg/mL L-glutamine and 10 mM HEPES pH 7.4. Cellsare subcultured every 2-3 days by trypsinization using 0.05% trypsin and0.02% EDTA in a calcium- and magnesium-free saline solution (Gibco BRL,Gaithersburg, Md.). To obtain well-spread single cells, cells are platedat a low density onto No. 1½ (18×18 mm) cover glasses in 100 mm culturedishes, and used 24-48 hours after plating.

Compound 30 is separately dissolved in DMSO to prepare a 1 mM to 10 mMdye stock solution. The stock solution is kept sealed in small aliquots,at −20° C. The stock solution is kept frozen at all times until use, andexposure to light is minimized. One aliquot of dye stock is taken fromthe freezer immediately before an experiment and thawed completely atroom temperature. The labeling solution is then prepared by adding thedye stock solution to fresh culture medium in an amount sufficient tomake final dye concentrations of 0.5-25 μM.

The optimal concentration of the dye for staining will vary dependingupon the application. Testing at least a tenfold range of concentrationis recommended. In general, long-term staining (more than about 3 days)or the use of rapidly dividing cells will require 5-25 μM dye. Less dye(0.5-5 μM) is needed for shorter experiments, such as viability assays.To maintain normal cellular physiology and reduce potential artifacts,the concentration of the dye should be kept as low as possible.

For cells in suspension, centrifuge the cells to pellet than andaspirate the supernatant. Resuspend the cells gently in prewarmedlabeling solution. Incubate the cells for 15-45 minutes under growthconditions appropriate for the particular cell type. Centrifuge thecells. For adherent cells, when the cells have reached the desiredconfluence, remove the medium from the dish and add the prewarmedlabeling solution. Incubate the cells for 15-45 minutes under growthconditions appropriate for the particular cell type.

Replace the labeling solution with fresh, prewarmed medium and incubatethe cultures for another 30 minutes at 37° C. During this time, thechloromethyl group of Compound 30 will undergo modification or will besecreted from the cell. The chloromethyl group reacts with thiols,probably in a glutathione S-transferase reaction. Therefore Compound 30is transformed into a cell-impermeant fluorescent dye-thioether adductthat can be fixed with aldahyde fixatives, permitting long-term samplestorage. Excess unconjugated dye passively diffuses to the extracellularmatrix.

The cells are attached to coverslips treated with BD-Cell-Tak (BecktonDickenson; Franklin Lakes, N.J.) and then washed with PBS. The labeledcells are then observed using a Zeiss Axioplan epifluorescencemicroscope equipped with an appropriate filter set.

Example 45 Preparation of a Tyramine Conjugate

To a solution of Compound 28 (0.1 mmol) in anhydrous DMF (2 mL) isslowly added 1 mL DMF solution of tyramine (0.22 mmol). The resultedmixture is stirred at room temperature for 5-8 h until the dye iscompletely consumed. The reaction solution is concentrated in vacuo, andpoured into ethyl acetate. The resulting precipitate is collected byfiltration and washed with ethyl acetate. The crude material is furtherpurified by HPLC to give the desired product.

Example 46 Cytotoxicity assay using Compound 32 (3H-phenoxazin-3-one,2,8-difluoro, 7-hydroxy-, 10-oxide)

HeLa and HepG2 cells are obtained from the American Type CultureCollection. The cells are cultured in Dulbecco's modified Eagle medium(DMEM) supplemented with 10% fetal bovine serum (Invitrogen) untilconfluent. Cells are harvested by trypsinization and their density isassessed by hemocytometer. Cells are diluted in medium to a density of1×10⁵ cells per mL. 200 μL (2×10⁴ cells) of cell suspension is added toeach well of a 96-well microplate. Cell samples are treated with variousdoses of cytotoxic test compounds (e.g. cisplatin 0.2-10 μg/mL) inserum-free medium for 2 hours during incubation in a humidifiedatmosphere at 37° C. and 5% CO₂. Compound 32 (10 μM) is then added toall cell samples including controls (no cytotoxic test compound added)followed by a further 2 hour incubation at 37° C. Fluorescencemeasurements on all samples is then performed at 37° C. using excitationat 530 nm and emission detection at 590 nm in a Victor² microplatereader (PerkinElmer Life Sciences). Cytotoxicity of the test compound isindicated by decreased fluorescence relative to the control samples.

Example 47 Preparation of a Phalloidin Dye-Conjugate

To aminophalloidin p-toluenesulfonate (3.5 mg, 4 μmol) and Compound 28(6.0 mg, 5 μmol) in DMF is added N,N-diisopropylethylamine (2 μL, 11μmol). The mixture is stirred at room temperature for 3 hours. To thisis added 7 mL of diethyl ether. The solid is collected bycentrifugation. The crude product is purified on SEPHADEX LH-20, elutingwith water to give the pure phalloidin conjugate.

Example 48 Preparation of a Drug Dye-Conjugate

A fluorescent dopamine D₂ antagonist is prepared as follows: To 10 mg ofN-(p-aminophenethyl)spiperone (Amlalky et al., FEBS LETT 176, 436(1984)), and 10 μL N,N-diisopropylethylamine in 1 mL of DMF is added 15mg of Compound 28. After 3 hours, the reaction mixture is poured into 5mL ether. The precipitate is centrifuged, then purified bychromatography on silica gel using 10-30% methanol in chloroform.

Example 49 Preparation of Protein Dye-Conjugates

A series of dye conjugates of goat anti-mouse IgG, streptavidin andother proteins, including R-phycoerythrin (R-PE) are prepared bystandard means (Haugland et al., METH. MOL. BIOL. 45, 205 (1995);Haugland, METH. MOL. BIOL. 45, 223 (1995); Haugland, METH. MOL. BIOL.45, 235 (1995)) using Compound 28.

A solution of the desired protein is prepared at 10 mg/mL in 0.1 Msodium bicarbonate. The labeling reagents are dissolved in DMF at 10mg/mL. Predetermined amounts of the labeling reagents are added to theprotein solutions with stirring. A molar ratio of 10 equivalents of dyeto 1 equivalent of protein is typical, though the optimal amount varieswith the particular labeling reagent, the protein being labeled and theprotein's concentration, and is determined empirically. The reactionmixture is incubated at room temperature for one hour, or on ice forseveral hours. The dye-protein conjugate is typically separated fromfree unreacted reagent by size-exclusion chromatography on BIO-RAD P-30resin equilibrated with PBS. The initial, protein-containing coloredband is collected and the degree of substitution is determined from theabsorbance at the absorbance maximum of each fluorophore, using theextinction coefficient of the free fluorophore.

Example 50 Labeling and Use of a Wheat Germ Agglutinin Dye-Conjugate

Wheat germ agglutinin (100 mg, EY Laboratories) is dissolved in 5 mLNaHCO₃, pH 8.3, containing 9 mg N-acetylglucosamine. To this is added 9mg of Compound 28. After 1 hour the solution is purified by gelfiltration. A degree of substitution of 2-3 dyes per molecule isdetermined from the absorption at 633 nm.

A 1 mg/mL stock solution of the resulting wheat germ agglutinin (WGA)conjugate (Compound 28) is prepared in 0.1 M sodium bicarbonate ˜pH 8.Staphylococcus aureus are cultured for 17 hours at 30° C. in TSB broth.Equal volumes of the TSB culture and a BSA solution (0.25% BSA+0.85%NaCl sterile filtered through 0.2 μM filter) are incubated at roomtemperature for 15 minutes. The BSA-bacterial suspension (200 μL) iscentrifuged for 2 minutes at 350×g, capturing the bacteria on a filtermembrane. The cells are resuspended in 90 μl of BSA solution and 10 μlof stain is added for 15 minutes. Following centrifugation, the bacteriaare resuspended in BSA solution, and an aliquot is trapped between aslide and a glass coverslip.

The bacteria are observed on a Nikon Diaphot epi-fluorescencemicroscope. Images are acquired using the Star-1 cooled CCD camera andthe software package supplied with the camera is used for data analysis.Two images are collected for each stain, each image having a 2 sec.exposure time. When used according to Sizemore et al. (U.S. Pat. No.5,137,810) the conjugate can distinguish between Gram positive and Gramnegative bacteria.

Example 51 Detection of Lipase Activity

Compound 4 is resuspended to 10 mM in dry DMSO. In 50 μl of MOPS buffer,pH 7.2, glyceryl triacetate and Triton X-100 is serially diluted acrossa 96-well microplate. To this serial dilution, 50 μl of Compound 4,lipase (porcine pancreas, Sigma L-0382), glycerokinase (Sigma, G-0774),glycerol 1-phosphate oxidase (Sigma, G-4388), horseradish peroxidase(Sigma, P-8250), and ATP in MOPS buffer, pH 7.2 is added. The finalconcentration in the well is 25 μM Compound 4, 500 Units/ml lipase, 0.5Unit/ml glycerokinase, 2 Units/ml glycerol 1-phosphate oxidase, 0.5Units/ml horseradish peroxidase, 0.5 mM ATP, zero to 228 μM glyceryltriacetate, and zero to 0.5% Triton X-100 in 50 mM MOPS buffer, pH 7.2.

This mixture is reacted for sixty minutes in the dark at 37° C., thenread in a fluorescence microplate reader. At that point, the resultingfluorescence is measured on a PerSeptive Biosystems CytoFluor 4000microtiter plate reader (Framingham, Mass.). The excitation filter is530 nm (+/−12.5 nm) and the emission filter 590 nm (+/−17.5 nm), with again setting of 40.

This reaction results in oxidation of Compound 4, and subsequentdetection by fluorescence. Compound 4 can be used to detecttriglycerides using the assay described above.

The preceding examples can be repeated with similar success bysubstituting the specifically described fluorogenic compounds of thepreceding examples with those generically and specifically described inthe forgoing description. One skilled in the art can easily ascertainthe essential characteristics of the present invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt to various usages andconditions.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto included within the spirit and purview of this application and areconsidered within the scope of the appended claims. All patents andpatent applications cited herein are hereby incorporated by reference intheir entirety for all purposes.

1-7. (canceled)
 8. An oxazine or thiazine dye compound having theformula:

wherein A is OH; Z is O; X is oxygen or sulfur; R² is hydrogen, halogen,substituted alkyl, unsubstituted alkyl, substituted alkoxy,unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio,substituted aryl, unsubstituted aryl, substituted heteroaryl,unsubstituted heteroaryl, sulfo, nitro, carboxyl, hydroxyl, a reactivegroup, substituted reactive group, a carrier molecule, substitutedcarrier molecule, a solid support or substituted solid support; R³ ishydrogen, halogen, unsubstituted alkyl, substituted alkoxy,unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio,substituted aryl, unsubstituted aryl, substituted heteroaryl,unsubstituted heteroaryl, sulfo, nitro, carboxyl, hydroxyl, a reactivegroup, substituted reactive group, a carrier molecule, substitutedcarrier molecule, a solid support or substituted solid support; R⁴ ishydrogen, halogen, substituted alkyl, unsubstituted alkyl, substitutedalkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstitutedalkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl,unsubstituted heteroaryl, sulfo, nitro, carboxyl, hydroxyl, a reactivegroup, substituted reactive group, a carrier molecule, substitutedcarrier molecule, a solid support or substituted solid support; R⁵ ishydrogen, halogen, substituted alkyl, unsubstituted alkyl, substitutedalkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstitutedalkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl,unsubstituted heteroaryl, sulfo, nitro, carboxyl, hydroxyl, a reactivegroup, substituted reactive group, a carrier molecule, substitutedcarrier molecule, a solid support or substituted solid support; R⁶ ishydrogen, halogen, substituted alkyl, unsubstituted alkyl, substitutedalkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstitutedalkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl,unsubstituted heteroaryl, sulfo, nitro, carboxyl, hydroxyl, a reactivegroup, substituted reactive group, a carrier molecule, substitutedcarrier molecule, a solid support or substituted solid support; R⁷ ishydrogen, halogen, substituted alkyl, unsubstituted alkyl, substitutedalkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstitutedalkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl,unsubstituted heteroaryl, sulfo, nitro, carboxyl, hydroxyl, a reactivegroup, substituted reactive group, a carrier molecule, substitutedcarrier molecule, a solid support or substituted solid support; or amember independently selected from R² in combination with R³; and R⁶ incombination with R⁷, together with the atoms to which they are joined,form a ring which is a 5-, 6- or 7-membered cycloalkyl, a substituted5-, 6- or 7-membered cycloalkyl, a 5-, 6- or 7-memberedheterocycloalkyl, a substituted 5-, 6- or 7-membered heterocycloalkyl, a5-, 6- or 7-membered aryl, a substituted 5-, 6- or 7-membered aryl, a5-, 6- or 7-membered heteroaryl, or a substituted 5-, 6- or 7-memberedheteroaryl; with the proviso that at least one member selected from R²,R³, R⁴, R⁵, R⁶ and R⁷ is fluorine.
 9. The compound according to claim 8,wherein (i) R³ and R⁶ are each fluorine and R², R⁴, R⁵ and R⁷ arehydrogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy,unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio,substituted aryl, unsubstituted aryl, substituted heteroaryl, orunsubstituted heteroaryl; or (ii) R⁴ and R⁵ are each fluorine and R²,R³, R⁶ and R⁷ are hydrogen, substituted alkyl, unsubstituted alkyl,substituted alkoxy, unsubstituted alkoxy, substituted alkylthio,unsubstituted alkylthio, substituted aryl, unsubstituted aryl,substituted heteroaryl, or unsubstituted heteroaryl; or (iii) R³, R⁴, R⁵and R⁶ are each fluorine and R² and R⁷ are hydrogen, substituted alkyl,unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy,substituted alkylthio, unsubstituted alkylthio, substituted aryl,unsubstituted aryl, substituted heteroaryl, or unsubstituted heteroaryl.10. A method for determining the presence or absence of peroxide in asample, wherein the method comprises: a) contacting the sample with afluorogenic compound having a structure of the formula:

wherein A is OH; E is OH; R¹ is methyl; X¹ is oxygen; X is oxygen; andeither: (i) R³ and R⁶ are each fluorine and R², R⁴, R⁵ and R⁷ arehydrogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy,unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio,substituted aryl, unsubstituted aryl, substituted heteroaryl, orunsubstituted heteroaryl; or (ii) R⁴ and R⁵ are each fluorine and R²,R³, R⁶ and R⁷ are hydrogen, substituted alkyl, unsubstituted alkyl,substituted alkoxy, unsubstituted alkoxy, substituted alkylthio,unsubstituted alkylthio, substituted aryl, unsubstituted aryl,substituted heteroaryl, or unsubstituted heteroaryl; or (iii) R³, R⁴, R⁵and R⁶ are each fluorine and R² and R⁷ are hydrogen, substituted alkyl,unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy,substituted alkylthio, unsubstituted alkylthio, substituted aryl,unsubstituted aryl, substituted heteroaryl, or unsubstituted heteroarylto prepare a labeled sample; b) incubating the labeled sample for asufficient amount of time to prepare an incubated sample, wherein theperoxide reacts with the compound in the presence of a peroxidase toproduce a fluorescent product; c) illuminating the incubated sample withan appropriate wavelength to prepare an illuminated sample; and d)observing the illuminated sample whereby the presence or absence of theperoxide in the sample is determined.
 11. The method according to claim10, wherein the peroxidase is horseradish peroxidase, myeloperoxidase orcyclooxygenase.
 12. A method for determining the presence or absence ofperoxide in a sample, wherein the method comprises: a) generatingperoxide from an enzymatic reaction wherein the enzymatic reaction isthe oxidation of a substrate by an oxidase to prepare a peroxidecontaining sample; b) contacting the peroxide containing sample with afluorogenic compound having a structure of the formula:

wherein A is OH; E is OH; R¹ is methyl; X¹ is oxygen; X is oxygen; andeither: (i) R³ and R⁶ are each fluorine and R², R⁴, R⁵ and R⁷ arehydrogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy,unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio,substituted aryl, unsubstituted aryl, substituted heteroaryl, orunsubstituted heteroaryl; or (ii) R⁴ and R⁵ are each fluorine and R²,R³, R⁶ and R⁷ are hydrogen, substituted alkyl, unsubstituted alkyl,substituted alkoxy, unsubstituted alkoxy, substituted alkylthio,unsubstituted alkylthio, substituted aryl, unsubstituted aryl,substituted heteroaryl, or unsubstituted heteroaryl; or (iii) R³, R⁴, R⁵and R⁶ are each fluorine and R² and R⁷ are hydrogen, substituted alkyl,unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy,substituted alkylthio, unsubstituted alkylthio, substituted aryl,unsubstituted aryl, substituted heteroaryl, or unsubstituted heteroarylto prepare a labeled sample, c) incubating the labeled sample for asufficient amount of time to prepare an incubated sample, wherein theperoxide reacts with the compound in the presence of a peroxidase toproduce a fluorescent product; d) illuminating the incubated sample withan appropriate wavelength to prepare an illuminated sample; and e)observing the illuminated sample whereby the presence or absence ofperoxide in the sample is determine. 13-20. (canceled)
 21. A compoundhaving a structure of the formula:

wherein A is OR⁸ or NR⁹R¹⁰ Z is O or N⁺R⁹R¹⁰; wherein R⁸ is hydrogen,substituted alkyl, unsubstituted alkyl, substituted heteroalkyl,unsubstituted heteroalkyl, substituted heterocycloalkyl, unsubstitutedheterocycloalkyl, substituted carboxyalkyl, unsubstituted carboxyalkyl,substituted sulfoalkyl, unsubstituted sulfoalkyl, substituted acyl,unsubstituted acyl, substituted haloalkyl, unsubstituted haloalkyl,substituted alkoxy, unsubstituted alkoxy, a reactive group, substitutedreactive group, carrier molecule, substituted carrier molecule, solidsupport or substituted solid support; R⁹ is hydrogen, substituted alkyl,unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl,substituted heterocycloalkyl, unsubstituted heterocycloalkyl,substituted carboxyalkyl, unsubstituted carboxyalkyl, substitutedsulfoalkyl, unsubstituted sulfoalkyl, substituted acyl, unsubstitutedacyl, substituted haloalkyl, unsubstituted haloalkyl, substitutedalkoxy, unsubstituted alkoxy, a reactive group, substituted reactivegroup, carrier molecule, substituted carrier molecule, solid support orsubstituted solid support; R¹⁰ is hydrogen, substituted alkyl,unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl,substituted heterocycloalkyl, unsubstituted heterocycloalkyl,substituted carboxyalkyl, unsubstituted carboxyalkyl, substitutedsulfoalkyl, unsubstituted sulfoalkyl, substituted acyl, unsubstitutedacyl, substituted haloalkyl, unsubstituted haloalkyl, substitutedalkoxy, unsubstituted alkoxy, a reactive group, substituted reactivegroup, carrier molecule, substituted carrier molecule, solid support orsubstituted solid support; or a member independently selected from R⁹ incombination with R¹⁰; R⁹ in combination with R³; R⁹ in combination withR⁶; R¹⁰ in combination with R⁴; and R¹⁰ in combination with R⁵ togetherwith the atoms to which they are joined, form a ring which is a 5-, 6-or 7-membered heterocycloalkyl, a substituted 5-, 6- or 7-memberedheterocycloalkyl, a 5-, 6- or 7-membered heteroaryl, or a substituted5-, 6- or 7-membered heteroaryl; X is oxygen or sulfur; and R² ishydrogen, halogen, substituted alkyl, unsubstituted alkyl, substitutedalkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstitutedalkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl,unsubstituted heteroaryl, sulfo, nitro, carboxyl, hydroxyl, a reactivegroup, substituted reactive group, a carrier molecule, substitutedcarrier molecule, a solid support or substituted solid support; R³ ishydrogen, halogen, unsubstituted alkyl, substituted alkoxy,unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio,substituted aryl, unsubstituted aryl, substituted heteroaryl,unsubstituted heteroaryl, sulfo, nitro, carboxyl, hydroxyl, a reactivegroup, substituted reactive group, a carrier molecule, substitutedcarrier molecule, a solid support or substituted solid support; R⁴ ishydrogen, halogen, substituted alkyl, unsubstituted alkyl, substitutedalkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstitutedalkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl,unsubstituted heteroaryl, sulfo, nitro, carboxyl, hydroxyl, a reactivegroup, substituted reactive group, a carrier molecule, substitutedcarrier molecule, a solid support or substituted solid support; R⁵ ishydrogen, halogen, substituted alkyl, unsubstituted alkyl, substitutedalkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstitutedalkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl,unsubstituted heteroaryl, sulfo, nitro, carboxyl, hydroxyl, a reactivegroup, substituted reactive group, a carrier molecule, substitutedcarrier molecule, a solid support or substituted solid support; R⁶ ishydrogen, halogen, substituted alkyl, unsubstituted alkyl, substitutedalkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstitutedalkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl,unsubstituted heteroaryl, sulfo, nitro, carboxyl, hydroxyl, a reactivegroup, substituted reactive group, a carrier molecule, substitutedcarrier molecule, a solid support or substituted solid support; R⁷ ishydrogen, halogen, substituted alkyl, unsubstituted alkyl, substitutedalkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstitutedalkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl,unsubstituted heteroaryl, sulfo, nitro, carboxyl, hydroxyl, a reactivegroup, substituted reactive group, a carrier molecule, substitutedcarrier molecule, a solid support or substituted solid support; or amember independently selected from R² in combination with R³; and R⁶ incombination with R⁷ together with the atoms to which they are joined,form a ring which is a 5-, 6- or 7-membered cycloalkyl, a substituted5-, 6- or 7-membered cycloalkyl, a 5-, 6- or 7-memberedheterocycloalkyl, a substituted 5-, 6- or 7-membered heterocycloalkyl, a5-, 6- or 7-membered aryl, a substituted 5-, 6- or 7-membered aryl, a5-, 6- or 7-membered heteroaryl, or a substituted 5-, 6- or 7-memberedheteroaryl; with the proviso that at least one member selected from R²,R³, R⁴, R⁵, R⁶ and R⁷ is fluorine.
 22. A method for detecting metabolicactivity in cells in a sample, wherein the method comprises: a)contacting the sample with a compound according to claim 21 to prepare alabeled sample; b) incubating the labeled sample for a sufficient amountof time to prepare an incubated sample, wherein the compound is capableof entering cells and being reduced to produce a fluorescent product; c)illuminating the incubated sample with an appropriate wavelength; and d)observing the illuminated sample whereby the metabolic activity isdetected and the resulting signal is proportional to the number ofviable cells present in the sample.
 23. A method for staining a sample,wherein the method comprises: a) contacting the sample with a compoundaccording to claim 8 to prepare a labeled sample; b) incubating thelabeled sample for a sufficient amount of time to prepare an incubatedsample; c) illuminating the incubated sample with an appropriatewavelength; and d) observing the illuminated sample whereby the sampleis stained.
 24. The method according to claim 10, wherein R³ and R⁶ areeach fluorine and R², R⁴, R⁵ and R⁷ are hydrogen, substituted alkyl,unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy,substituted alkylthio, unsubstituted alkylthio, substituted aryl,unsubstituted aryl, substituted heteroaryl, or unsubstituted heteroaryl.25. The method of claim 24, wherein R², R⁴, R⁵ and R⁷ are hydrogen. 26.The method of claim 10, wherein R⁴ and R⁵ are each fluorine and R², R³,R⁶ and R⁷ are hydrogen, substituted alkyl, unsubstituted alkyl,substituted alkoxy, unsubstituted alkoxy, substituted alkylthio,unsubstituted alkylthio, substituted aryl, unsubstituted aryl,substituted heteroaryl, or unsubstituted heteroaryl.
 27. The method ofclaim 26, wherein R², R³, R⁶ and R⁷ are hydrogen.
 28. The method ofclaim 1, wherein R³, R⁴, R⁵ and R⁶ are each fluorine and R² and R⁷ arehydrogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy,unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio,substituted aryl, unsubstituted aryl, substituted heteroaryl, orunsubstituted heteroaryl.